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    Name:
    Mesenchymal Stem Cell Osteogenic Differentiation Medium
    Description:
    Mesenchymal Stem Cell Osteogenic Differentiation Medium Ready to use
    Catalog Number:
    C-28013
    Price:
    None
    Applications:
    Mesenchymal Stem Cell Osteogenic Differentiation Medium was designed for the directed differentiation of mesenchymal stem cells (MSC) from bone marrow, the umbilical cord matrix (Wharton´s Jelly) and adipose tissue into osteogenic lineages. Recommended for:Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM)Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC)Human Mesenchymal Stem Cells from Adipose Tissue (hMSC-AT)
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    millipore mscs
    Mesenchymal Stem Cell Osteogenic Differentiation Medium
    Mesenchymal Stem Cell Osteogenic Differentiation Medium Ready to use
    https://www.bioz.com/result/mscs/product/millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mscs - by Bioz Stars, 2021-09
    86/100 stars

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    1) Product Images from "Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome, et al. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome"

    Article Title: Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome, et al. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome

    Journal: Aging Cell

    doi: 10.1111/acel.13152

    Increased expression of SASP factors in Z24 −/− MSCs negatively impacts muscle stem cell function. (a) Gastrocnemius (GM) skeletal muscle tissues were harvested from 5‐month‐old Z24 −/− and wild‐type (WT) mice. Immunostaining analysis of PDGFR‐α and CD68 showed increased number of PDGFR‐α + MSCs and CD68 + macrophages. Scale bar = 100 µm. (b) Quantification of CD68 + cells is shown. (c) Immunostaining analysis of dystrophin and Pax7 showed a decreased number of Pax7 + muscle stem cell in Z24 −/− mice. (d) Quantification of the ratio of the PDGFR‐α + cells to Pax7 + cells is shown. (d) Immunostaining analysis of PDGFR‐α and Pax7 demonstrating a close interaction between these two types of cells in stem cell niche. White arrow indicates a Pax7 + cell; orange arrow indicates a PDGFR‐α + cell. Scale bar = 50 µm. (e) Quantification of mRNA level of PDGFR‐α in muscles is shown. (f) Immunostaining analysis of PDGFR‐α and Pax7 to show their relative localization at stem cell niche. Scale bar = 10µm. (g) Immunostaining analysis of PDGFR‐α in mesenchymal stem/stromal cells (MSCs) from WT mice and Z24 −/− mice. Scale bar = 100 µm. (h) qPCR results of mRNA from WT MSC and Z24 −/− MSCs. (i) Treatment of WT muscle progenitor cells (MPCs) with conditioned medium from WT or Z24 −/− MSCs to check the impact on myogenesis potential [the formation of fast‐myosin heavy chain (f‐MHC)‐positive myotubes], and level of DNA damage (γ‐H2AX). Quantitation of cells positive with f‐MHC or γ‐H2AX is shown. Scale bar = 100 µm. (j) qPCR results of mRNA from WT MPCs treated with conditioned medium from WT or Z24 −/− MSCs. Data are shown as mean ± standard error. N ≥ 6. * indicates p
    Figure Legend Snippet: Increased expression of SASP factors in Z24 −/− MSCs negatively impacts muscle stem cell function. (a) Gastrocnemius (GM) skeletal muscle tissues were harvested from 5‐month‐old Z24 −/− and wild‐type (WT) mice. Immunostaining analysis of PDGFR‐α and CD68 showed increased number of PDGFR‐α + MSCs and CD68 + macrophages. Scale bar = 100 µm. (b) Quantification of CD68 + cells is shown. (c) Immunostaining analysis of dystrophin and Pax7 showed a decreased number of Pax7 + muscle stem cell in Z24 −/− mice. (d) Quantification of the ratio of the PDGFR‐α + cells to Pax7 + cells is shown. (d) Immunostaining analysis of PDGFR‐α and Pax7 demonstrating a close interaction between these two types of cells in stem cell niche. White arrow indicates a Pax7 + cell; orange arrow indicates a PDGFR‐α + cell. Scale bar = 50 µm. (e) Quantification of mRNA level of PDGFR‐α in muscles is shown. (f) Immunostaining analysis of PDGFR‐α and Pax7 to show their relative localization at stem cell niche. Scale bar = 10µm. (g) Immunostaining analysis of PDGFR‐α in mesenchymal stem/stromal cells (MSCs) from WT mice and Z24 −/− mice. Scale bar = 100 µm. (h) qPCR results of mRNA from WT MSC and Z24 −/− MSCs. (i) Treatment of WT muscle progenitor cells (MPCs) with conditioned medium from WT or Z24 −/− MSCs to check the impact on myogenesis potential [the formation of fast‐myosin heavy chain (f‐MHC)‐positive myotubes], and level of DNA damage (γ‐H2AX). Quantitation of cells positive with f‐MHC or γ‐H2AX is shown. Scale bar = 100 µm. (j) qPCR results of mRNA from WT MPCs treated with conditioned medium from WT or Z24 −/− MSCs. Data are shown as mean ± standard error. N ≥ 6. * indicates p

    Techniques Used: Expressing, Cell Function Assay, Mouse Assay, Immunostaining, Real-time Polymerase Chain Reaction, Quantitation Assay

    Z24 −/− MSCs display increased senescent phenotypes, and enhanced F‐actin polymerization and cytoskeletal stiffness is directly associated with increased and nuclear blebbing. MSCs isolated from the skeletal muscle of WT and Z24 −/− mice were compared. (a) Immunostaining analysis of γ‐H2AX, p21 Cip1 , and lamin A/C was performed, as well as SA‐β‐Gal staining for senescence. Quantitation of γ‐H2AX + cells, SA‐β‐Gal + cells, p21 + cells, and cells with nuclear blebbing is shown. Scale bar = 30µm. (b) Immunostaining analysis and quantification of H3K9me3. The increased level of H3K9me3 (red) in the micronuclei in contrast to nucleus indicates the loss of heterochromatin from nucleus to micronuclei (arrows). Scale bar = 2.5 µm. (c) Staining of F‐actin with Alexa Fluor 488 Phalloidin and quantification of F‐actin polymerization. Scale bar = 20 µm. (d–g). Testing of cytoplasm stiffness using a Bruker AFM probe. H. The cytoplasm stiffness (kPa) calculated by NanoScope analysis. (i) Immunostaining analysis of lamin A/C and F‐actin in WT and Z24 −/− MSCs, showing higher level of F‐actin and nuclear blebbing in same Z24 −/− cell (arrow). Scale bar = 50 µm. (j) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs. Quantitation of nuclear blebbing is shown. The number of cells with nuclear blebbing was compared between cells with top 30% of F‐actin intensity (Actin‐high) and cells with bottom 30% of F‐actin intensity (Actin‐low). Scale bar = 30 µm. (k) Immunostaining analysis of lamin A/C and F‐actin to observe the effect of treatment of Z24 −/− MSCs with F‐actin stabilizing JPK (200 nM) or F‐actin depolymerizing CyD (100 ng/ml) for 48 hr. Quantitation of nuclear blebbing is shown. Scale bar = 15 µm. Arrows: nuclear blebbing. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Z24 −/− MSCs display increased senescent phenotypes, and enhanced F‐actin polymerization and cytoskeletal stiffness is directly associated with increased and nuclear blebbing. MSCs isolated from the skeletal muscle of WT and Z24 −/− mice were compared. (a) Immunostaining analysis of γ‐H2AX, p21 Cip1 , and lamin A/C was performed, as well as SA‐β‐Gal staining for senescence. Quantitation of γ‐H2AX + cells, SA‐β‐Gal + cells, p21 + cells, and cells with nuclear blebbing is shown. Scale bar = 30µm. (b) Immunostaining analysis and quantification of H3K9me3. The increased level of H3K9me3 (red) in the micronuclei in contrast to nucleus indicates the loss of heterochromatin from nucleus to micronuclei (arrows). Scale bar = 2.5 µm. (c) Staining of F‐actin with Alexa Fluor 488 Phalloidin and quantification of F‐actin polymerization. Scale bar = 20 µm. (d–g). Testing of cytoplasm stiffness using a Bruker AFM probe. H. The cytoplasm stiffness (kPa) calculated by NanoScope analysis. (i) Immunostaining analysis of lamin A/C and F‐actin in WT and Z24 −/− MSCs, showing higher level of F‐actin and nuclear blebbing in same Z24 −/− cell (arrow). Scale bar = 50 µm. (j) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs. Quantitation of nuclear blebbing is shown. The number of cells with nuclear blebbing was compared between cells with top 30% of F‐actin intensity (Actin‐high) and cells with bottom 30% of F‐actin intensity (Actin‐low). Scale bar = 30 µm. (k) Immunostaining analysis of lamin A/C and F‐actin to observe the effect of treatment of Z24 −/− MSCs with F‐actin stabilizing JPK (200 nM) or F‐actin depolymerizing CyD (100 ng/ml) for 48 hr. Quantitation of nuclear blebbing is shown. Scale bar = 15 µm. Arrows: nuclear blebbing. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Isolation, Mouse Assay, Immunostaining, Staining, Quantitation Assay

    Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Activation Assay, Over Expression, Immunostaining, Activity Assay, Expressing, Western Blot, Mouse Assay, Transfection, Plasmid Preparation, Staining

    Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Staining, Quantitation Assay, Western Blot

    RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction, Staining

    2) Product Images from "Mesenchymal stem cell senescence alleviates their intrinsic and seno-suppressive paracrine properties contributing to osteoarthritis development"

    Article Title: Mesenchymal stem cell senescence alleviates their intrinsic and seno-suppressive paracrine properties contributing to osteoarthritis development

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102379

    Senescence modulates MSCs intrinsic properties. ( A ) Beta-galactosidase staining in human MSCs at day 14 after DNA damaged-induced senescence (Senescent) or not (Proliferative). Data are the mean ± SEM (n=5); ****=p
    Figure Legend Snippet: Senescence modulates MSCs intrinsic properties. ( A ) Beta-galactosidase staining in human MSCs at day 14 after DNA damaged-induced senescence (Senescent) or not (Proliferative). Data are the mean ± SEM (n=5); ****=p

    Techniques Used: Staining

    3) Product Images from "Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells"

    Article Title: Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-12-67

    Morphological change of MSCs with or without the exposure to CART . Particular changes in morphology happened to MSCs with the exposure to CART. Similar to the situation of the bFGF/EGF-treated group, several MSCs incubated with CART for 3 days became shorter and nucleus-convergent. They subsequently evolved to display round cell bodies with long axons in 6 days. In the control group, mesenchmal stem cells nearly kept stable in the appearance within the 6 days of, observation (scale bar = 50 uM).
    Figure Legend Snippet: Morphological change of MSCs with or without the exposure to CART . Particular changes in morphology happened to MSCs with the exposure to CART. Similar to the situation of the bFGF/EGF-treated group, several MSCs incubated with CART for 3 days became shorter and nucleus-convergent. They subsequently evolved to display round cell bodies with long axons in 6 days. In the control group, mesenchmal stem cells nearly kept stable in the appearance within the 6 days of, observation (scale bar = 50 uM).

    Techniques Used: Incubation

    Differential spectrum of MSCs determined by Immunofluorescence . Nestin, GFAP, MAP-2 and NeuN were involved to detect neural progenitors, glial cells, and mature neurons. In the CART treated group (A), the percentage of Nestin (green) positive cells occupied 25.4 ± 2.1% in Day 3 and 47.1 ± 1.9% in Day 6. (B) The NeuN (green) positive rate was 32.1 ± 2.3% in 3 days and 40.3 ± 2.7% in 6 days. The differentiation ratio resembled that of bFGF/EGF. (C) MAP-2 (red) was detected in 30.8 ± 4.7% and 41.2 ± 3.1% of all cells in Day 3 and Day 6. (D) GFAP (green) ranks 20.5 ± 2.5% in 3 days and 21.3 ± 2.2% in 6 days. The converted rate resembled that of bFGF/EGF. The untreated group was used as a control..
    Figure Legend Snippet: Differential spectrum of MSCs determined by Immunofluorescence . Nestin, GFAP, MAP-2 and NeuN were involved to detect neural progenitors, glial cells, and mature neurons. In the CART treated group (A), the percentage of Nestin (green) positive cells occupied 25.4 ± 2.1% in Day 3 and 47.1 ± 1.9% in Day 6. (B) The NeuN (green) positive rate was 32.1 ± 2.3% in 3 days and 40.3 ± 2.7% in 6 days. The differentiation ratio resembled that of bFGF/EGF. (C) MAP-2 (red) was detected in 30.8 ± 4.7% and 41.2 ± 3.1% of all cells in Day 3 and Day 6. (D) GFAP (green) ranks 20.5 ± 2.5% in 3 days and 21.3 ± 2.2% in 6 days. The converted rate resembled that of bFGF/EGF. The untreated group was used as a control..

    Techniques Used: Immunofluorescence

    Founctional test of the differentiated MSCs . Cholinergic neurons and dopaminergic neurons were investigated by double labeled immunofluorescence with the antibodies against ChAT (green) and NeuN as well as TH (green) and NeuN (red) with the purpose of finding founctional neurons. NeuN positive cells co-expressed with ChAT (A) or TH (B) in the CART-treated group and the bFGF/EGF-incubated group. (C) The transferred neurons exhibited cytoplasmic dark blue praticles and light blue nucleus by Nissl stain. MSCs devoid of CART displayed dark blue nucleus and light blue cytoplasm (scale bar = 50 uM).
    Figure Legend Snippet: Founctional test of the differentiated MSCs . Cholinergic neurons and dopaminergic neurons were investigated by double labeled immunofluorescence with the antibodies against ChAT (green) and NeuN as well as TH (green) and NeuN (red) with the purpose of finding founctional neurons. NeuN positive cells co-expressed with ChAT (A) or TH (B) in the CART-treated group and the bFGF/EGF-incubated group. (C) The transferred neurons exhibited cytoplasmic dark blue praticles and light blue nucleus by Nissl stain. MSCs devoid of CART displayed dark blue nucleus and light blue cytoplasm (scale bar = 50 uM).

    Techniques Used: Labeling, Immunofluorescence, Incubation, Staining

    4) Product Images from "The Fate and Distribution of Autologous Bone Marrow Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs"

    Article Title: The Fate and Distribution of Autologous Bone Marrow Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs

    Journal: Stem Cells International

    doi: 10.1155/2016/8616143

    The distribution of MSCs with intra-arterial infusion in femoral head and vital organs. Immunohistochemical staining of BrdU-positive MSCs in different tissues at eight weeks after transplantation was observed under a light microscope. (a) Femoral head. (b) Heart; (c) liver; (d) spleen; (e) lung; (f) kidney; (g) stomach; (h) gallbladder; (i) small bowel; (j) pancreas; (k) prostate; (l) testicle. Scale bar 20 μm . Particularly, BrdU strongly positive MSCs were significantly located in the tissues of the kidney, gallbladder, and liver.
    Figure Legend Snippet: The distribution of MSCs with intra-arterial infusion in femoral head and vital organs. Immunohistochemical staining of BrdU-positive MSCs in different tissues at eight weeks after transplantation was observed under a light microscope. (a) Femoral head. (b) Heart; (c) liver; (d) spleen; (e) lung; (f) kidney; (g) stomach; (h) gallbladder; (i) small bowel; (j) pancreas; (k) prostate; (l) testicle. Scale bar 20 μm . Particularly, BrdU strongly positive MSCs were significantly located in the tissues of the kidney, gallbladder, and liver.

    Techniques Used: Immunohistochemistry, Staining, Transplantation Assay, Light Microscopy

    Osteogenic and adipogenic differentiation of MSCs with intra-arterial infusion in necrotic field of femoral head. BrdU (green) and osteocalcin (red) were analyzed by double-immunofluorescent analysis in the necrotic region of MSC-transplanted femoral heads. (a) DAPI (blue); (b) BrdU (red); (c) osteocalcin (green); (d) an overlay of (a), (b), and (c); (e) DAPI (blue); (f) BrdU (red); (g) PPAR- γ (green); (h) an overlay of (e), (f), and (g). Most of the BrdU-positive MSCs in the necrotic region of MSC-transplanted femoral heads costained together with osteocalcin but a few cells costained together with PPAR- γ . Yellow arrow shows osteoblasts differentiated from MSCs, red arrow shows adipocytes differentiated from MSCs, and grey arrow shows BrdU-positive cells which did not differentiate to lipocytes. Scale bar 200 μm . BrdU + /osteocalcin + cells in MSCs group (76.42 ± 10.14%) were significantly higher than BrdU + /PPAR- γ + cells over total BrdU + cells (6.36 ± 4.41%) after eight-week treatment ( p
    Figure Legend Snippet: Osteogenic and adipogenic differentiation of MSCs with intra-arterial infusion in necrotic field of femoral head. BrdU (green) and osteocalcin (red) were analyzed by double-immunofluorescent analysis in the necrotic region of MSC-transplanted femoral heads. (a) DAPI (blue); (b) BrdU (red); (c) osteocalcin (green); (d) an overlay of (a), (b), and (c); (e) DAPI (blue); (f) BrdU (red); (g) PPAR- γ (green); (h) an overlay of (e), (f), and (g). Most of the BrdU-positive MSCs in the necrotic region of MSC-transplanted femoral heads costained together with osteocalcin but a few cells costained together with PPAR- γ . Yellow arrow shows osteoblasts differentiated from MSCs, red arrow shows adipocytes differentiated from MSCs, and grey arrow shows BrdU-positive cells which did not differentiate to lipocytes. Scale bar 200 μm . BrdU + /osteocalcin + cells in MSCs group (76.42 ± 10.14%) were significantly higher than BrdU + /PPAR- γ + cells over total BrdU + cells (6.36 ± 4.41%) after eight-week treatment ( p

    Techniques Used:

    Evaluation of MSCs and BrdU-labeling efficacy. Primary cultured MSCs exhibit typical elongated, fibroblast-like morphology or large, flattened shape (a). Scale bar 200 μm . MSCs in third passage were positive for BrdU, as immunofluorescence stained with BrdU (b). Scale bar 100 μm . Flow cytometry analysis of the surface markers of bone marrow mononuclear cells: CD29 (99.91%), CD90 (97.52%), CD11b (6.63%), and CD45 (7.07%) (c).
    Figure Legend Snippet: Evaluation of MSCs and BrdU-labeling efficacy. Primary cultured MSCs exhibit typical elongated, fibroblast-like morphology or large, flattened shape (a). Scale bar 200 μm . MSCs in third passage were positive for BrdU, as immunofluorescence stained with BrdU (b). Scale bar 100 μm . Flow cytometry analysis of the surface markers of bone marrow mononuclear cells: CD29 (99.91%), CD90 (97.52%), CD11b (6.63%), and CD45 (7.07%) (c).

    Techniques Used: Labeling, Cell Culture, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    5) Product Images from "Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome, et al. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome"

    Article Title: Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome, et al. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome

    Journal: Aging Cell

    doi: 10.1111/acel.13152

    Increased expression of SASP factors in Z24 −/− MSCs negatively impacts muscle stem cell function. (a) Gastrocnemius (GM) skeletal muscle tissues were harvested from 5‐month‐old Z24 −/− and wild‐type (WT) mice. Immunostaining analysis of PDGFR‐α and CD68 showed increased number of PDGFR‐α + MSCs and CD68 + macrophages. Scale bar = 100 µm. (b) Quantification of CD68 + cells is shown. (c) Immunostaining analysis of dystrophin and Pax7 showed a decreased number of Pax7 + muscle stem cell in Z24 −/− mice. (d) Quantification of the ratio of the PDGFR‐α + cells to Pax7 + cells is shown. (d) Immunostaining analysis of PDGFR‐α and Pax7 demonstrating a close interaction between these two types of cells in stem cell niche. White arrow indicates a Pax7 + cell; orange arrow indicates a PDGFR‐α + cell. Scale bar = 50 µm. (e) Quantification of mRNA level of PDGFR‐α in muscles is shown. (f) Immunostaining analysis of PDGFR‐α and Pax7 to show their relative localization at stem cell niche. Scale bar = 10µm. (g) Immunostaining analysis of PDGFR‐α in mesenchymal stem/stromal cells (MSCs) from WT mice and Z24 −/− mice. Scale bar = 100 µm. (h) qPCR results of mRNA from WT MSC and Z24 −/− MSCs. (i) Treatment of WT muscle progenitor cells (MPCs) with conditioned medium from WT or Z24 −/− MSCs to check the impact on myogenesis potential [the formation of fast‐myosin heavy chain (f‐MHC)‐positive myotubes], and level of DNA damage (γ‐H2AX). Quantitation of cells positive with f‐MHC or γ‐H2AX is shown. Scale bar = 100 µm. (j) qPCR results of mRNA from WT MPCs treated with conditioned medium from WT or Z24 −/− MSCs. Data are shown as mean ± standard error. N ≥ 6. * indicates p
    Figure Legend Snippet: Increased expression of SASP factors in Z24 −/− MSCs negatively impacts muscle stem cell function. (a) Gastrocnemius (GM) skeletal muscle tissues were harvested from 5‐month‐old Z24 −/− and wild‐type (WT) mice. Immunostaining analysis of PDGFR‐α and CD68 showed increased number of PDGFR‐α + MSCs and CD68 + macrophages. Scale bar = 100 µm. (b) Quantification of CD68 + cells is shown. (c) Immunostaining analysis of dystrophin and Pax7 showed a decreased number of Pax7 + muscle stem cell in Z24 −/− mice. (d) Quantification of the ratio of the PDGFR‐α + cells to Pax7 + cells is shown. (d) Immunostaining analysis of PDGFR‐α and Pax7 demonstrating a close interaction between these two types of cells in stem cell niche. White arrow indicates a Pax7 + cell; orange arrow indicates a PDGFR‐α + cell. Scale bar = 50 µm. (e) Quantification of mRNA level of PDGFR‐α in muscles is shown. (f) Immunostaining analysis of PDGFR‐α and Pax7 to show their relative localization at stem cell niche. Scale bar = 10µm. (g) Immunostaining analysis of PDGFR‐α in mesenchymal stem/stromal cells (MSCs) from WT mice and Z24 −/− mice. Scale bar = 100 µm. (h) qPCR results of mRNA from WT MSC and Z24 −/− MSCs. (i) Treatment of WT muscle progenitor cells (MPCs) with conditioned medium from WT or Z24 −/− MSCs to check the impact on myogenesis potential [the formation of fast‐myosin heavy chain (f‐MHC)‐positive myotubes], and level of DNA damage (γ‐H2AX). Quantitation of cells positive with f‐MHC or γ‐H2AX is shown. Scale bar = 100 µm. (j) qPCR results of mRNA from WT MPCs treated with conditioned medium from WT or Z24 −/− MSCs. Data are shown as mean ± standard error. N ≥ 6. * indicates p

    Techniques Used: Expressing, Cell Function Assay, Mouse Assay, Immunostaining, Real-time Polymerase Chain Reaction, Quantitation Assay

    Z24 −/− MSCs display increased senescent phenotypes, and enhanced F‐actin polymerization and cytoskeletal stiffness is directly associated with increased and nuclear blebbing. MSCs isolated from the skeletal muscle of WT and Z24 −/− mice were compared. (a) Immunostaining analysis of γ‐H2AX, p21 Cip1 , and lamin A/C was performed, as well as SA‐β‐Gal staining for senescence. Quantitation of γ‐H2AX + cells, SA‐β‐Gal + cells, p21 + cells, and cells with nuclear blebbing is shown. Scale bar = 30µm. (b) Immunostaining analysis and quantification of H3K9me3. The increased level of H3K9me3 (red) in the micronuclei in contrast to nucleus indicates the loss of heterochromatin from nucleus to micronuclei (arrows). Scale bar = 2.5 µm. (c) Staining of F‐actin with Alexa Fluor 488 Phalloidin and quantification of F‐actin polymerization. Scale bar = 20 µm. (d–g). Testing of cytoplasm stiffness using a Bruker AFM probe. H. The cytoplasm stiffness (kPa) calculated by NanoScope analysis. (i) Immunostaining analysis of lamin A/C and F‐actin in WT and Z24 −/− MSCs, showing higher level of F‐actin and nuclear blebbing in same Z24 −/− cell (arrow). Scale bar = 50 µm. (j) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs. Quantitation of nuclear blebbing is shown. The number of cells with nuclear blebbing was compared between cells with top 30% of F‐actin intensity (Actin‐high) and cells with bottom 30% of F‐actin intensity (Actin‐low). Scale bar = 30 µm. (k) Immunostaining analysis of lamin A/C and F‐actin to observe the effect of treatment of Z24 −/− MSCs with F‐actin stabilizing JPK (200 nM) or F‐actin depolymerizing CyD (100 ng/ml) for 48 hr. Quantitation of nuclear blebbing is shown. Scale bar = 15 µm. Arrows: nuclear blebbing. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Z24 −/− MSCs display increased senescent phenotypes, and enhanced F‐actin polymerization and cytoskeletal stiffness is directly associated with increased and nuclear blebbing. MSCs isolated from the skeletal muscle of WT and Z24 −/− mice were compared. (a) Immunostaining analysis of γ‐H2AX, p21 Cip1 , and lamin A/C was performed, as well as SA‐β‐Gal staining for senescence. Quantitation of γ‐H2AX + cells, SA‐β‐Gal + cells, p21 + cells, and cells with nuclear blebbing is shown. Scale bar = 30µm. (b) Immunostaining analysis and quantification of H3K9me3. The increased level of H3K9me3 (red) in the micronuclei in contrast to nucleus indicates the loss of heterochromatin from nucleus to micronuclei (arrows). Scale bar = 2.5 µm. (c) Staining of F‐actin with Alexa Fluor 488 Phalloidin and quantification of F‐actin polymerization. Scale bar = 20 µm. (d–g). Testing of cytoplasm stiffness using a Bruker AFM probe. H. The cytoplasm stiffness (kPa) calculated by NanoScope analysis. (i) Immunostaining analysis of lamin A/C and F‐actin in WT and Z24 −/− MSCs, showing higher level of F‐actin and nuclear blebbing in same Z24 −/− cell (arrow). Scale bar = 50 µm. (j) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs. Quantitation of nuclear blebbing is shown. The number of cells with nuclear blebbing was compared between cells with top 30% of F‐actin intensity (Actin‐high) and cells with bottom 30% of F‐actin intensity (Actin‐low). Scale bar = 30 µm. (k) Immunostaining analysis of lamin A/C and F‐actin to observe the effect of treatment of Z24 −/− MSCs with F‐actin stabilizing JPK (200 nM) or F‐actin depolymerizing CyD (100 ng/ml) for 48 hr. Quantitation of nuclear blebbing is shown. Scale bar = 15 µm. Arrows: nuclear blebbing. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Isolation, Mouse Assay, Immunostaining, Staining, Quantitation Assay

    Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Activation Assay, Over Expression, Immunostaining, Activity Assay, Expressing, Western Blot, Mouse Assay, Transfection, Plasmid Preparation, Staining

    Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Staining, Quantitation Assay, Western Blot

    RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction, Staining

    Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Increased RhoA activation in Z24 −/− MSCs, and effect of RhoA over‐expression on Sun2 and nuclear blebbing in WT MSCs. (a) Immunostaining analysis of RhoA and F‐actin in WT and Z24 −/− MSCs. Scale bar = 30 µm. (b) Quantification of RhoA + cells is shown. (c) Quantification of RhoA activity is shown. (d) Immunostaining analysis of RhoA and lamin A/C in Z24 −/− MSCs. Arrows: cells with higher RhoA expression and nuclear blebbing. Scale bar = 5 µm. (e) Quantification of nuclear blebbing in RhoA + and RhoA‐ Z24 −/− MSCs is shown. (f, g) Western blot analysis and quantification of RhoA in WT and Z24 −/− MSCs, with GAPDH as loading control. (h) Immunostaining analysis of RhoA + cells and CD68 + inflammatory cells in skeletal muscle of Z24 −/− mice. (i, j) Western blot analysis and quantification of RhoA in muscle tissues from WT and Z24 −/− mice, with GAPDH as loading control. (k) WT MSCs were transfected with a plasmid carrying constitutively active RhoA‐GFP and stained for F‐actin. Scale bar = 5 µm. (l) Immunostaining analysis of Sun2 to check Sun2 and nuclear blebbing in RhoA‐GFP transfected WT MSCs. Yellow arrows: cells with RhoA‐GFP; red arrows: cells without RhoA‐GFP. Scale bar = 5 µm. (m) Quantification nuclear blebbing (RhoA‐GFP‐ V.S. RhoA‐GFP + cells) is shown. (n) Immunostaining analysis of Sun2 and lamin A/C in Z24 −/− MSCs. Scale bar = 10µm. (o) Quantification of Sun2 in Z24 −/− MSCs without or without nuclear blebbing is shown. (p) Quantification of Sun2 in WT and Z24 −/− MSCs is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Activation Assay, Over Expression, Immunostaining, Activity Assay, Expressing, Western Blot, Mouse Assay, Transfection, Plasmid Preparation, Staining

    Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: Effect of inhibition of RhoA/ROCK signaling or Sun2 expression in Z24 −/− MSCs. (a) Immunostaining analysis of lamin A/C and F‐actin in Z24 −/− MSCs treated with Rho activator II or RhoA/ROCK inhibitor Y‐27632. Quantification of nuclear blebbing and F‐actin is shown. Scale bar = 5 µm. (b) Immunostaining analysis and quantification of γ‐H2AX and SA‐β‐Gal staining in Z24 −/− MSCs treated with Y‐27632. Quantification of γ‐H2AX + or SA‐β‐Gal + cells is shown. Scale bar = 100 µm. (c) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs treated with Y‐27632 or C3 transferase (C3). Quantification of Sun2 with or without RhoA inhibition is shown. Scale bar = 3 µm. (d) Immunostaining analysis of Sun1 and Sun2 in nuclear and micronuclei of Z24 −/− MSCs. Scale bar = 3 µm. (e) Quantitation of Sun1 and Sun2 protein level in micronuclei of Z24 −/− MSCs is shown. (f) Demonstration of perinuclear actin cap stress fiber. (g) Immunostaining analysis of Sun2 and F‐actin in Z24 −/− MSCs with or without Sun2 SiRNA treatment. Scale bar = 3 µm. (h) Quantitation of Sun2 and nuclear blebbing is shown. (i) Quantification of F‐actin level is shown. (j) Western blot analysis of Sun1 and Sun2 in Z24 −/− MSCs and Z24 −/− MSCs treated with Y‐27632 or Sun2 SiRNA. (k) Quantitation of Sun1 and Sun2 in western blot result is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Staining, Quantitation Assay, Western Blot

    RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p
    Figure Legend Snippet: RhoA inhibition in Z24 −/− MSCs represses micronuclei/cytoplasmic DNA‐induced innate immune response, reduces SASP expression, and rescues senescent phenotypes. (a) Immunostaining analysis of lamin A/C and cGAS showed that there is positive cGAS deposition at the micronuclei formed in Z24 −/− MSCs (arrows). Scale bar = 3 µm. (b) Western blot analysis and quantification of proteins related to the cGAS‐Sting signaling (cGAS, phosphor‐p65, phosphor‐TBK1) in WT MSCs, Z24 −/− MSCs, and Z24 −/− MSCs treated with Y‐27632. (c) qPCR analysis of interferon‐1β (IFN‐1β) expression. (d) qPCR analysis of the expression of SASP and senescent‐associated genes in Z24 −/− MSCs with or without Y‐27632 treatment. (e) Osteogenesis assay and adipogenesis assay of Z24 −/− MSCs with or without Y‐27632 treatment. Osteogenic potential was examined with ALP staining of osteogenic cells, and adipogenic potential was examined with AdipoRed staining of lipid in adipogenic cells. Scale bar = 30 µm. (f) Quantification of ALP or AdipoRed is shown. (g) Immunostaining analysis of lamin A/C in Z24 −/− MPCs treated with conditioned medium from Z24 −/− MSCs with or without Y‐27632 pretreatment. Arrows indicate cells with nuclear blebbing. Scale bar = 50 µm. (h) Quantification of myotube number and nuclear blebbing is shown. N ≥ 6. “*” at bar charts indicates p

    Techniques Used: Inhibition, Expressing, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction, Staining

    6) Product Images from "The Effects of Synthetic Oligopeptide Derived from Enamel Matrix Derivative on Cell Proliferation and Osteoblastic Differentiation of Human Mesenchymal Stem Cells"

    Article Title: The Effects of Synthetic Oligopeptide Derived from Enamel Matrix Derivative on Cell Proliferation and Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150814026

    Effect of PD98059 on the production of PIP and OCN induced by SP. After the MSCs reached confluence, the culture medium was replaced with osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM) and the cells were cultured for 7 ( A ) or 14 ( B ) days. * p
    Figure Legend Snippet: Effect of PD98059 on the production of PIP and OCN induced by SP. After the MSCs reached confluence, the culture medium was replaced with osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM) and the cells were cultured for 7 ( A ) or 14 ( B ) days. * p

    Techniques Used: Cell Culture

    Effect of SP on mineralization. ( A ) Confluent MSCs were stained with Alizarin Red after 7 or 14 days of cultivation in osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM). Scale bar = 100 µm; ( B , C ) The extracellular calcium deposition was measured at 7 or 14 days. * p
    Figure Legend Snippet: Effect of SP on mineralization. ( A ) Confluent MSCs were stained with Alizarin Red after 7 or 14 days of cultivation in osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM). Scale bar = 100 µm; ( B , C ) The extracellular calcium deposition was measured at 7 or 14 days. * p

    Techniques Used: Staining

    Effect of PD98059 on SP-induced cell proliferation. MSCs were seeded in 96-well plates at 2 × 10 3 cells/well in normal culture medium. After a 24 h culture for cell adherence, MSCs were cultured in normal culture medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor PD98059 (10 µM). Cell proliferation was measured on days 1, 3, 5 and 7. * p
    Figure Legend Snippet: Effect of PD98059 on SP-induced cell proliferation. MSCs were seeded in 96-well plates at 2 × 10 3 cells/well in normal culture medium. After a 24 h culture for cell adherence, MSCs were cultured in normal culture medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor PD98059 (10 µM). Cell proliferation was measured on days 1, 3, 5 and 7. * p

    Techniques Used: Cell Culture

    Effect of PD98059 on ALP staining and ALP activity induced by SP. ( A ) Confluent MSCs were stained using an ALP staining kit after 7 or 14 days of cultivation in osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM). Scale bar = 100 µm; ( B , C ) ALP activity was measured at 7 or 14 days. To normalize ALP activity, the amount of ALP was normalized to the amount of DNA. * p
    Figure Legend Snippet: Effect of PD98059 on ALP staining and ALP activity induced by SP. ( A ) Confluent MSCs were stained using an ALP staining kit after 7 or 14 days of cultivation in osteogenic medium containing SP (0 or 10 ng/mL), or SP (10 ng/mL) with the ERK 1/2 inhibitor, PD98059 (10 µM). Scale bar = 100 µm; ( B , C ) ALP activity was measured at 7 or 14 days. To normalize ALP activity, the amount of ALP was normalized to the amount of DNA. * p

    Techniques Used: ALP Assay, Staining, Activity Assay

    7) Product Images from "Suboptimal Level of Bone‐Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy"

    Article Title: Suboptimal Level of Bone‐Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy

    Journal: Hepatology Communications

    doi: 10.1002/hep4.1234

    Images of BM biopsy in Cirrhosis and Control cases. (A) Panel I. Representative Images of cirrhosis and control BM (left) showing the osteoblast cells and bar graph (right) showing number of osteoblast (mean ± SE) in cirrhosis and control BM (H E). Panel II. Cirrhosis and control BM (left) showing the osteocytes and bar graph (right) showing number of osteocytes (mean ± SE) in cirrhosis and control BM (H E). Panel III. Alcian blue‐stained chondroblasts in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of chondroblasts (mean ± SE) in cirrhosis and control BM. (B) Panel I. Representing osteocalcin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of osteocalcin+ cells in cirrhosis and control BM. Panel II. Osteocalcin+ area (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing percentage osteocalcin+ area in cirrhosis and control BM. Panel III. Showing osteonectin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing osteonectin in cirrhosis and control BM (C) Nestin+ BM MSCs in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of these cells in cirrhosis and control BM. (D) CD169+ BM macrophage (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of CD169+ cells in cirrhosis and control BM. (All image magnification ×200, area 1.3 mm 2 ). Abbreviations: BM, bone marrow; H E, hematoxylin and eosin.
    Figure Legend Snippet: Images of BM biopsy in Cirrhosis and Control cases. (A) Panel I. Representative Images of cirrhosis and control BM (left) showing the osteoblast cells and bar graph (right) showing number of osteoblast (mean ± SE) in cirrhosis and control BM (H E). Panel II. Cirrhosis and control BM (left) showing the osteocytes and bar graph (right) showing number of osteocytes (mean ± SE) in cirrhosis and control BM (H E). Panel III. Alcian blue‐stained chondroblasts in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of chondroblasts (mean ± SE) in cirrhosis and control BM. (B) Panel I. Representing osteocalcin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of osteocalcin+ cells in cirrhosis and control BM. Panel II. Osteocalcin+ area (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing percentage osteocalcin+ area in cirrhosis and control BM. Panel III. Showing osteonectin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing osteonectin in cirrhosis and control BM (C) Nestin+ BM MSCs in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of these cells in cirrhosis and control BM. (D) CD169+ BM macrophage (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of CD169+ cells in cirrhosis and control BM. (All image magnification ×200, area 1.3 mm 2 ). Abbreviations: BM, bone marrow; H E, hematoxylin and eosin.

    Techniques Used: Staining

    Distribution graphs. (A) Panel I. Osteoblasts. Panel II. Osteocytes. Panel III. Chondroblasts. (B) Panel I. Osteocalcin+ cells. Panel II. Percentage osteocalcin+ area. Panel III. Osteonectin+ cells. (C) Nestin+ MSCs. (D) CD169+ BM macrophages (all expressed in mean ± SE) in BM sections of patients with cirrhosis with normal bone density, osteopenia, and osteoporosis; evaluated by T score on bone densitometry scan.
    Figure Legend Snippet: Distribution graphs. (A) Panel I. Osteoblasts. Panel II. Osteocytes. Panel III. Chondroblasts. (B) Panel I. Osteocalcin+ cells. Panel II. Percentage osteocalcin+ area. Panel III. Osteonectin+ cells. (C) Nestin+ MSCs. (D) CD169+ BM macrophages (all expressed in mean ± SE) in BM sections of patients with cirrhosis with normal bone density, osteopenia, and osteoporosis; evaluated by T score on bone densitometry scan.

    Techniques Used:

    8) Product Images from "Olfactory Ensheathing Cells Do Not Exhibit Unique Migratory or Axonal Growth-Promoting Properties after Spinal Cord Injury"

    Article Title: Olfactory Ensheathing Cells Do Not Exhibit Unique Migratory or Axonal Growth-Promoting Properties after Spinal Cord Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3264-06.2006

    Other cell types form tracts to the lesion site when injected acutely after injury. A–F , GFP and GFAP double-fluorescent immunolabeling indicates that fibroblasts also form tracts extending from the injection site (IS) to the lesion cavity (Les) when injected into dorsal white matter, 1 mm rostral or caudal to the lesion site. The time course of cellular distribution and filling of the lesion cavity entirely parallels that of OECs and MSCs (data not shown). G , Similar responses are observed after injection of bone marrow stromal cells (example of cells 3 h after injection is shown). Scale bars: A , C , E , G , 350 μm; B , D , F , 50 μm.
    Figure Legend Snippet: Other cell types form tracts to the lesion site when injected acutely after injury. A–F , GFP and GFAP double-fluorescent immunolabeling indicates that fibroblasts also form tracts extending from the injection site (IS) to the lesion cavity (Les) when injected into dorsal white matter, 1 mm rostral or caudal to the lesion site. The time course of cellular distribution and filling of the lesion cavity entirely parallels that of OECs and MSCs (data not shown). G , Similar responses are observed after injection of bone marrow stromal cells (example of cells 3 h after injection is shown). Scale bars: A , C , E , G , 350 μm; B , D , F , 50 μm.

    Techniques Used: Injection, Immunolabeling

    9) Product Images from "Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells"

    Article Title: Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

    Journal: Stem Cells International

    doi: 10.1155/2015/498328

    TNF α negatively regulates EC differentiation: the effect of TNF α on VEGF-A-stimulated differentiation of MSCs into ECs was examined. TNF α R and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3-4) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p
    Figure Legend Snippet: TNF α negatively regulates EC differentiation: the effect of TNF α on VEGF-A-stimulated differentiation of MSCs into ECs was examined. TNF α R and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3-4) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS, Angiogenesis Assay

    Ang II positively regulates VEGF-A-mediated EC differentiation: the effect of Ang II on VEGF-A-stimulated differentiation of MSCs into ECs was examined. AT2R and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 5-6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p
    Figure Legend Snippet: Ang II positively regulates VEGF-A-mediated EC differentiation: the effect of Ang II on VEGF-A-stimulated differentiation of MSCs into ECs was examined. AT2R and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 5-6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS, Angiogenesis Assay

    IL-6 negatively regulates EC differentiation: the effect of IL-6 on VEGF-A-stimulated differentiation of MSCs into ECs was examined. IL-6R and VEGFR-2 mRNA expression was analyzed by RT-PCR and normalized to GAPDH ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis and normalized to GAPDH ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3–6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p
    Figure Legend Snippet: IL-6 negatively regulates EC differentiation: the effect of IL-6 on VEGF-A-stimulated differentiation of MSCs into ECs was examined. IL-6R and VEGFR-2 mRNA expression was analyzed by RT-PCR and normalized to GAPDH ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis and normalized to GAPDH ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3–6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS, Angiogenesis Assay

    Characterization of BM-MSCs: detailed FACS characterization revealed that MSCs at P3 to P5 stained negatively for CD14 and CD45 but expressed surface markers that are indicative of MSC lineage, including CD44, CD73, and CD90 (a). Isolated MSCs also exhibited fibroblastoid-like morphology (b). Naïve BM-MSCs demonstrated the capacity to differentiate in osteogenic, chondrogenic, and adipogenic lineages. Alizarin red showed staining of calcium deposits in MSCs differentiated into the osteogenic lineage. Alcian/Safranin O blue showed staining of peptidoglycans characteristic of differentiation into the chondrogenic lineage. Oil Red O showed staining of the lipids and triglycerides, indicating differentiation into the adipogenic lineage. Each image shown is representative of independent experiments performed with BM-MSC cultures derived from separate microswine ( n = 3). FACS analysis performed for EC markers on naïve MSCs (c), MSCs treated with VEGF-A (d), and HUVECs (e). Each grid shown is representative of independent experiments performed with cultures derived from bone marrow of separate microswine ( n = 3–6).
    Figure Legend Snippet: Characterization of BM-MSCs: detailed FACS characterization revealed that MSCs at P3 to P5 stained negatively for CD14 and CD45 but expressed surface markers that are indicative of MSC lineage, including CD44, CD73, and CD90 (a). Isolated MSCs also exhibited fibroblastoid-like morphology (b). Naïve BM-MSCs demonstrated the capacity to differentiate in osteogenic, chondrogenic, and adipogenic lineages. Alizarin red showed staining of calcium deposits in MSCs differentiated into the osteogenic lineage. Alcian/Safranin O blue showed staining of peptidoglycans characteristic of differentiation into the chondrogenic lineage. Oil Red O showed staining of the lipids and triglycerides, indicating differentiation into the adipogenic lineage. Each image shown is representative of independent experiments performed with BM-MSC cultures derived from separate microswine ( n = 3). FACS analysis performed for EC markers on naïve MSCs (c), MSCs treated with VEGF-A (d), and HUVECs (e). Each grid shown is representative of independent experiments performed with cultures derived from bone marrow of separate microswine ( n = 3–6).

    Techniques Used: FACS, Staining, Isolation, Derivative Assay

    Ang II counteracts IL-6 or TNF α inhibition of EC differentiation: the combined effects of Ang II, IL-6, and TNF α on VEGF-A-stimulated differentiation of MSCs into ECs were examined. TNF α R, IL-6R, AT2R, and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3-4) ((c)–(h)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(i)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p
    Figure Legend Snippet: Ang II counteracts IL-6 or TNF α inhibition of EC differentiation: the combined effects of Ang II, IL-6, and TNF α on VEGF-A-stimulated differentiation of MSCs into ECs were examined. TNF α R, IL-6R, AT2R, and VEGFR-2 mRNA expression was analyzed by RT-PCR ( n = 3) (a). Sox18 protein levels were measured by Western blot analysis ( n = 3) (b). Expression of EC markers was determined by FACS analysis, and a representative grid is shown ( n = 3-4) ((c)–(h)). Endothelial tube formation was examined using an angiogenesis assay ( n = 3) ((d)–(i)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗ p

    Techniques Used: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS, Angiogenesis Assay

    10) Product Images from "Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells"

    Article Title: Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7031

    OAd-infected MSC carriers invade tumor spheroids MSCs growing as a monolayer in 24-well plates were stained with the red fluorescent dye PKH26; 2 μM dye solution (provided by the manufacturer) was added per well. MSCs were infected with Ad5/3-Luc (Luc), Ad5/3-Δ19K-.Luc (Δ19K-) or Ad5/3-TRAIL (TRAIL) at a titer of 2000 TCID 50 24 h later. Two hours after viral infection, the MSCs were covered with a 1:1:1 mixture of Matrigel, collagen and methylcellulose in DMEM medium supplemented with 2% FCS. Immediately afterward, tumor spheroids prepared from the primary, PDA cell line PaCaDD in passage 10 or from the established PDA cell line MIAPaCa-2 were seeded on top of the gel layer. The cells were co-incubated for 16 h to allow invasion of the MSCs into the spheroids through the gel layer. The spheroids were then transferred into new culture plates using a pipette and cultivated until 42 h after infection. Invasion was evaluated by detection of adenoviral capsid protein (green) by staining with a specific antibody and double immunofluorescence microscopy. Bright field (left), fluorescence (right: Adenoviral capsid protein: green; MSCs: red; Merge: yellow). Representative images at 400× magnification are shown.
    Figure Legend Snippet: OAd-infected MSC carriers invade tumor spheroids MSCs growing as a monolayer in 24-well plates were stained with the red fluorescent dye PKH26; 2 μM dye solution (provided by the manufacturer) was added per well. MSCs were infected with Ad5/3-Luc (Luc), Ad5/3-Δ19K-.Luc (Δ19K-) or Ad5/3-TRAIL (TRAIL) at a titer of 2000 TCID 50 24 h later. Two hours after viral infection, the MSCs were covered with a 1:1:1 mixture of Matrigel, collagen and methylcellulose in DMEM medium supplemented with 2% FCS. Immediately afterward, tumor spheroids prepared from the primary, PDA cell line PaCaDD in passage 10 or from the established PDA cell line MIAPaCa-2 were seeded on top of the gel layer. The cells were co-incubated for 16 h to allow invasion of the MSCs into the spheroids through the gel layer. The spheroids were then transferred into new culture plates using a pipette and cultivated until 42 h after infection. Invasion was evaluated by detection of adenoviral capsid protein (green) by staining with a specific antibody and double immunofluorescence microscopy. Bright field (left), fluorescence (right: Adenoviral capsid protein: green; MSCs: red; Merge: yellow). Representative images at 400× magnification are shown.

    Techniques Used: Infection, Staining, Incubation, Transferring, Immunofluorescence, Microscopy, Fluorescence

    11) Product Images from "Chemokine Receptors Expression in MSCs: Comparative Analysis in Different Sources and Passages"

    Article Title: Chemokine Receptors Expression in MSCs: Comparative Analysis in Different Sources and Passages

    Journal: Tissue Engineering and Regenerative Medicine

    doi: 10.1007/s13770-017-0069-7

    Migration ability of MSCs from three different sources toward SDF-1α in P2 and P3. A Cell migration of ICBM-, Ad- and RIA-MSCs was analyzed using a Boyden chamber assay in P2. B Cell migration of ICBM-, Ad- and RIA-MSCs was analyzed using a Boyden chamber assay in P3. Fluorescent images show representative of DAPI-stained cells ( upper panel ). Statistical analysis revealed that the migration capacity of Ad-MSCs was significantly higher than ICBM and RIA-MSCs in P2, but not in P3 ( lower panel ). * p
    Figure Legend Snippet: Migration ability of MSCs from three different sources toward SDF-1α in P2 and P3. A Cell migration of ICBM-, Ad- and RIA-MSCs was analyzed using a Boyden chamber assay in P2. B Cell migration of ICBM-, Ad- and RIA-MSCs was analyzed using a Boyden chamber assay in P3. Fluorescent images show representative of DAPI-stained cells ( upper panel ). Statistical analysis revealed that the migration capacity of Ad-MSCs was significantly higher than ICBM and RIA-MSCs in P2, but not in P3 ( lower panel ). * p

    Techniques Used: Migration, Boyden Chamber Assay, Staining

    The expression pattern for cell surface markers of MSCs from ICBM, Ad, RIA and HDF cells. A Flow cytometry analysis was done to profile characteristic markers of MSCs. ICBM-, Ad-, RIA-MSCs have shown the same immunophenotype but not with HDF as a control. B Flow cytometry results for negative surface markers of MSCs were negative similar to HDF. C Cumulative data of FACS analysis of MSCs from ICBM, Ad, RIA and HDF cells for several surface markers. ICBM Iliac crest bone marrow, Ad Adipose derived, RIA Reamer-irrigator-aspirator, HDF Human dermal fibroblast
    Figure Legend Snippet: The expression pattern for cell surface markers of MSCs from ICBM, Ad, RIA and HDF cells. A Flow cytometry analysis was done to profile characteristic markers of MSCs. ICBM-, Ad-, RIA-MSCs have shown the same immunophenotype but not with HDF as a control. B Flow cytometry results for negative surface markers of MSCs were negative similar to HDF. C Cumulative data of FACS analysis of MSCs from ICBM, Ad, RIA and HDF cells for several surface markers. ICBM Iliac crest bone marrow, Ad Adipose derived, RIA Reamer-irrigator-aspirator, HDF Human dermal fibroblast

    Techniques Used: Expressing, Flow Cytometry, Cytometry, FACS, Derivative Assay

    Differentiation capacities of MSCs populations toward osteoblast and adipocyte. A – D Spindle shaped fibroblastic-like cells in the primary culture of human MSCs derived from ICBM, Ad, and RIA. Human dermal fibroblast (HDF) is as control. E – H Osteogenic medium was used to MSCs and HDF cells induction at P2 toward osteoblasts for 21 days. MSCs after differentiation have produced calcified extracellular matrix that stained by Alizarin Red S reagent as evident for osteogenesis in comparison to HDF. I – L Additionally, osteogensis was confirmed by ALP assay of osteoblasts compared with HDF cells. M – P Also, Oil Red O staining was performed to staining of lipid droplets in order to prove adipogenic differentiation of MSCs, but not HDF, that were cultured in adipogenic induction medium. ICBM iliac crest bone marrow, Ad Adipose, RIA Reamer-irrigator-aspirator
    Figure Legend Snippet: Differentiation capacities of MSCs populations toward osteoblast and adipocyte. A – D Spindle shaped fibroblastic-like cells in the primary culture of human MSCs derived from ICBM, Ad, and RIA. Human dermal fibroblast (HDF) is as control. E – H Osteogenic medium was used to MSCs and HDF cells induction at P2 toward osteoblasts for 21 days. MSCs after differentiation have produced calcified extracellular matrix that stained by Alizarin Red S reagent as evident for osteogenesis in comparison to HDF. I – L Additionally, osteogensis was confirmed by ALP assay of osteoblasts compared with HDF cells. M – P Also, Oil Red O staining was performed to staining of lipid droplets in order to prove adipogenic differentiation of MSCs, but not HDF, that were cultured in adipogenic induction medium. ICBM iliac crest bone marrow, Ad Adipose, RIA Reamer-irrigator-aspirator

    Techniques Used: Derivative Assay, Produced, Staining, ALP Assay, Cell Culture

    The expression pattern for different chemokine receptors from ICBM, Ad- and RIA-MSCs using RT-PCR and Real time RT-PCR in P2 and P3. A The expression of CCR1 , CCR7 , CXCR2 , CXCR4 , CXCR6 and CX3CR1 in MSCs at P2. B The expression of CXCR4 and CXCR6 in MSCs at P3. C The expression of CCR1 , CCR7 , CXCR2 , and CX3CR1 in MSCs at P3. A – C CXCR4 and CXCR2 mRNA levels in RIA MSCs was undetectable in P2 and P3. The β - actin (ACTB) housekeeping gene product was used to as an endogenous reference. Human peripheral blood mononuclear cells (PBMCs) were used to a positive control. * p
    Figure Legend Snippet: The expression pattern for different chemokine receptors from ICBM, Ad- and RIA-MSCs using RT-PCR and Real time RT-PCR in P2 and P3. A The expression of CCR1 , CCR7 , CXCR2 , CXCR4 , CXCR6 and CX3CR1 in MSCs at P2. B The expression of CXCR4 and CXCR6 in MSCs at P3. C The expression of CCR1 , CCR7 , CXCR2 , and CX3CR1 in MSCs at P3. A – C CXCR4 and CXCR2 mRNA levels in RIA MSCs was undetectable in P2 and P3. The β - actin (ACTB) housekeeping gene product was used to as an endogenous reference. Human peripheral blood mononuclear cells (PBMCs) were used to a positive control. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control

    Morphology and population doubling times in three sources of MSCs. A Spindle shape fibroblast-like form was observed in all three sources at P0. B The population doubling time (PDT) of the ICBM-, Ad- and RIA-MSCs were approximately 5.1, 4.4 and 2.4 days, respectively. The PDT among the 6 passages had no significant change
    Figure Legend Snippet: Morphology and population doubling times in three sources of MSCs. A Spindle shape fibroblast-like form was observed in all three sources at P0. B The population doubling time (PDT) of the ICBM-, Ad- and RIA-MSCs were approximately 5.1, 4.4 and 2.4 days, respectively. The PDT among the 6 passages had no significant change

    Techniques Used:

    12) Product Images from "A Specific Subpopulation of Mesenchymal Stromal Cell Carriers Overrides Melanoma Resistance to an Oncolytic Adenovirus"

    Article Title: A Specific Subpopulation of Mesenchymal Stromal Cell Carriers Overrides Melanoma Resistance to an Oncolytic Adenovirus

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2011.0643

    Detection of fluorescence emission within the tumor architecture composed by CMDiI+ MO-MSCs and tumor melanoma cells (A375N) injected into nude mice. When MO-MSCs are preincubated with 1,10 Phenathroline there is a decrease in the migration into the inner
    Figure Legend Snippet: Detection of fluorescence emission within the tumor architecture composed by CMDiI+ MO-MSCs and tumor melanoma cells (A375N) injected into nude mice. When MO-MSCs are preincubated with 1,10 Phenathroline there is a decrease in the migration into the inner

    Techniques Used: Fluorescence, Injection, Mouse Assay, Migration

    13) Product Images from "Mesenchymal stem cells express serine protease inhibitor to evade the host immune response"

    Article Title: Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

    Journal: Blood

    doi: 10.1182/blood-2010-06-287979

    Longevity assessment of MSCs from WT and SPI6 −/− mice . (A) Schematic showing the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector (HRST-hGH) generated by transient transfection of 5 plasmids (pHDM-Hgpm2, pMD-Tat,
    Figure Legend Snippet: Longevity assessment of MSCs from WT and SPI6 −/− mice . (A) Schematic showing the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector (HRST-hGH) generated by transient transfection of 5 plasmids (pHDM-Hgpm2, pMD-Tat,

    Techniques Used: Mouse Assay, Plasmid Preparation, Generated, Transfection

    14) Product Images from "Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis"

    Article Title: Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.5b00294

    Emission maxima of selected MscS mutants in DDM and reconstituted into membrane bilayers. Six Trp mutants were purified and reconstituted into DOPC bilayers and the emission spectra recorded as described in Experimental Procedures . (A) The red-shift of the emission maxima can be seen in particular for residues in the hydrophobic section of the pore. (B) The positions of the residues are shown on the closed structure 2OAU. (C) Emission spectra of DDM-solubilized samples (black) and samples reconstituted into DOPC (red) are compared.
    Figure Legend Snippet: Emission maxima of selected MscS mutants in DDM and reconstituted into membrane bilayers. Six Trp mutants were purified and reconstituted into DOPC bilayers and the emission spectra recorded as described in Experimental Procedures . (A) The red-shift of the emission maxima can be seen in particular for residues in the hydrophobic section of the pore. (B) The positions of the residues are shown on the closed structure 2OAU. (C) Emission spectra of DDM-solubilized samples (black) and samples reconstituted into DOPC (red) are compared.

    Techniques Used: Purification

    Stability of MscS tryptophan mutants. (A) Western blot of E. coli MscS Trp mutants in which 15 μg of membrane protein was loaded on a 4 to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and after development Western blotting with antibody specific for the His 6 tag (see Experimental Procedures ). (B) Membrane proteins (30 μg) were solubilized in PBS (pH 7.4) containing 1% DDM, 2.8 M urea, and 1 mM EDTA and prepared for BN-PAGE as described in Experimental Procedures . Samples were separated on Novex 4 to 16% Bis-Tris gradient native gels (Invitrogen) and proteins detected by Western blot as described for panel A. The positions of heptameric and monomeric MscS with associated lipids and detergent are indicated.
    Figure Legend Snippet: Stability of MscS tryptophan mutants. (A) Western blot of E. coli MscS Trp mutants in which 15 μg of membrane protein was loaded on a 4 to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and after development Western blotting with antibody specific for the His 6 tag (see Experimental Procedures ). (B) Membrane proteins (30 μg) were solubilized in PBS (pH 7.4) containing 1% DDM, 2.8 M urea, and 1 mM EDTA and prepared for BN-PAGE as described in Experimental Procedures . Samples were separated on Novex 4 to 16% Bis-Tris gradient native gels (Invitrogen) and proteins detected by Western blot as described for panel A. The positions of heptameric and monomeric MscS with associated lipids and detergent are indicated.

    Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

    15) Product Images from "Simulated Microgravity Suppresses Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting Oxidative Phosphorylation"

    Article Title: Simulated Microgravity Suppresses Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting Oxidative Phosphorylation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21249747

    SMG inhibits osteogenesis and OXPHOS in MSCs after exposure to SMG. ( a ) Gene expression of ALP, OCN, RUNX2 after 72 h. ( b ) Relative ALP activity after 72 h. ( c ) Representative images of ALP staining after 7 day. ( d ) Gene expression of PGC-1α after 72 h. ( e ) Relative mtDNA copy number levels after 72 h. ( f ) Cells were stained with MitoTracker Green for mitochondrial mass and assessed by flow cytometry after 72 h. ( g ) Relative OCR levels after 72 h. For each group, the values are the mean ± SEM from three representative independent experiments. Control: NG control. * p
    Figure Legend Snippet: SMG inhibits osteogenesis and OXPHOS in MSCs after exposure to SMG. ( a ) Gene expression of ALP, OCN, RUNX2 after 72 h. ( b ) Relative ALP activity after 72 h. ( c ) Representative images of ALP staining after 7 day. ( d ) Gene expression of PGC-1α after 72 h. ( e ) Relative mtDNA copy number levels after 72 h. ( f ) Cells were stained with MitoTracker Green for mitochondrial mass and assessed by flow cytometry after 72 h. ( g ) Relative OCR levels after 72 h. For each group, the values are the mean ± SEM from three representative independent experiments. Control: NG control. * p

    Techniques Used: Expressing, Activity Assay, Staining, Pyrolysis Gas Chromatography, Flow Cytometry

    Enhancing OPXHOS via Sirt1 recovers osteogenesis of MSCs after exposure to SMG with resveratrol treatment. ( a ) Gene expression of ALP, OCN, and RUNX2 after 72 h. ( b ) Relative ALP activity after 72 h. ( c ) Representative images of ALP staining after 7 day. For each group, the values are the mean ± SEM from three representative independent experiments. Control—solvent control (DMSO). * p
    Figure Legend Snippet: Enhancing OPXHOS via Sirt1 recovers osteogenesis of MSCs after exposure to SMG with resveratrol treatment. ( a ) Gene expression of ALP, OCN, and RUNX2 after 72 h. ( b ) Relative ALP activity after 72 h. ( c ) Representative images of ALP staining after 7 day. For each group, the values are the mean ± SEM from three representative independent experiments. Control—solvent control (DMSO). * p

    Techniques Used: Expressing, Activity Assay, Staining

    16) Product Images from "Platelet-derived growth factor BB enhances osteogenesis of adipose-derived but not bone marrow-derived mesenchymal stromal/stem cells"

    Article Title: Platelet-derived growth factor BB enhances osteogenesis of adipose-derived but not bone marrow-derived mesenchymal stromal/stem cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2060

    Loss-of-function experiment for the effect of exogenous PDGF-BB. siRNA against the receptor PDGFRβ was delivered to MSCs and ASCs using a reducible poly[β-amino ester] vehicle. Knockdown of receptor relative to a scrambled control was
    Figure Legend Snippet: Loss-of-function experiment for the effect of exogenous PDGF-BB. siRNA against the receptor PDGFRβ was delivered to MSCs and ASCs using a reducible poly[β-amino ester] vehicle. Knockdown of receptor relative to a scrambled control was

    Techniques Used:

    Gene expression of MSCs and ASCs under the effect of exogenous PDGF-BB. Gene expression analysis of the osteogenic genes Runx2, osteocalcin, osteonectin, and collagen-I via real-time polymerase chain reaction showed that exogenous PDGF-BB under control
    Figure Legend Snippet: Gene expression of MSCs and ASCs under the effect of exogenous PDGF-BB. Gene expression analysis of the osteogenic genes Runx2, osteocalcin, osteonectin, and collagen-I via real-time polymerase chain reaction showed that exogenous PDGF-BB under control

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    MSC and ASC mineralization under the effect of exogenous PDGF-BB. MSCs and ASCs were cultured under control (−), control (+), osteogenic (−), and osteogenic (+) conditions for three weeks. Staining after three weeks of culture revealed
    Figure Legend Snippet: MSC and ASC mineralization under the effect of exogenous PDGF-BB. MSCs and ASCs were cultured under control (−), control (+), osteogenic (−), and osteogenic (+) conditions for three weeks. Staining after three weeks of culture revealed

    Techniques Used: Cell Culture, Staining

    17) Product Images from "Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform"

    Article Title: Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0034-3

    DRG neurons have an impact on Cx43 and N-cadherin expression in MSCs during osteoblast differentiation Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Subcellular distribution of Cx43 and N-cadherin in MSCs was evaluated at 4 and 7 days of coculture by IF using antibodies directed against Cx43 and N-cadherin coupled to Alexa Fluor ® 594 (red), and DAPI (nuclei; blue) under a confocal microscope. Scale bar = 100 μm. b Cx43 and N-cadherin expression in MSCs was analyzed at 4 and 7 days of coculture by WB and depicted as a relative ratio to the respective loading control α-tubulin normalized to the monoculture value on day 4. The results represent three independent experiments.
    Figure Legend Snippet: DRG neurons have an impact on Cx43 and N-cadherin expression in MSCs during osteoblast differentiation Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Subcellular distribution of Cx43 and N-cadherin in MSCs was evaluated at 4 and 7 days of coculture by IF using antibodies directed against Cx43 and N-cadherin coupled to Alexa Fluor ® 594 (red), and DAPI (nuclei; blue) under a confocal microscope. Scale bar = 100 μm. b Cx43 and N-cadherin expression in MSCs was analyzed at 4 and 7 days of coculture by WB and depicted as a relative ratio to the respective loading control α-tubulin normalized to the monoculture value on day 4. The results represent three independent experiments.

    Techniques Used: Expressing, Derivative Assay, Incubation, Microscopy, Western Blot

    Role of DRG neurons in different phases of osteoblast differentiation from MSCs DRG neurons promote the osteogenic differentiation potential of MSCs by regulating the canonical/β-catenin Wnt signaling pathway and expression of osteoblast-related genes/proteins in MSCs.
    Figure Legend Snippet: Role of DRG neurons in different phases of osteoblast differentiation from MSCs DRG neurons promote the osteogenic differentiation potential of MSCs by regulating the canonical/β-catenin Wnt signaling pathway and expression of osteoblast-related genes/proteins in MSCs.

    Techniques Used: Expressing

    DRG neurons induce cytoplasmic accumulation of β-catenin and its translocation into the nucleus in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Subcellular distribution of β-catenin in MSCs was evaluated at 4 and 7 days of coculture by IF using antibodies directed against β-catenin coupled to Alexa Fluor ® 488 (green), and DAPI (nuclei; blue) under a confocal microscope. Scale bar = 100 μm. b–d The level of fluorescence for β-catenin was measured at 4 and 7 days of coculture by using the ImageJ software in each MSC and normalized to the monoculture value on day 4. Data expressed as median, 25 and 75 percentiles, min/max. ( n ) indicates the total number of cells counted for each group. e Expression profile of Ctnnb1 in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture value on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p
    Figure Legend Snippet: DRG neurons induce cytoplasmic accumulation of β-catenin and its translocation into the nucleus in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Subcellular distribution of β-catenin in MSCs was evaluated at 4 and 7 days of coculture by IF using antibodies directed against β-catenin coupled to Alexa Fluor ® 488 (green), and DAPI (nuclei; blue) under a confocal microscope. Scale bar = 100 μm. b–d The level of fluorescence for β-catenin was measured at 4 and 7 days of coculture by using the ImageJ software in each MSC and normalized to the monoculture value on day 4. Data expressed as median, 25 and 75 percentiles, min/max. ( n ) indicates the total number of cells counted for each group. e Expression profile of Ctnnb1 in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture value on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p

    Techniques Used: Translocation Assay, Derivative Assay, Incubation, Microscopy, Fluorescence, Software, Expressing, Quantitative RT-PCR

    DRG neurons lead to colocalization between β-catenin and Lef1 into the nucleus, where together regulate the expression of Wnt-responsive genes in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 4 and 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Nuclear colocalization of active-β-catenin and Lef1 in MSCs was evaluated at 4 and 7 days of coculture by IF with antibodies directed against active-β-catenin coupled to Alexa Fluor ® 488 (green), Lef1 coupled to Alexa Fluor ® 594 (red), and DAPI (nuclei; blue) under a confocal microscope. Arrows point to nuclear colocalization. Scale bar = 100 and 20 μm inset images. b and c Expression profile of Tnfrsf11b and Ccn1/Cyr61 in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR, and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture value on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p
    Figure Legend Snippet: DRG neurons lead to colocalization between β-catenin and Lef1 into the nucleus, where together regulate the expression of Wnt-responsive genes in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 4 and 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a Nuclear colocalization of active-β-catenin and Lef1 in MSCs was evaluated at 4 and 7 days of coculture by IF with antibodies directed against active-β-catenin coupled to Alexa Fluor ® 488 (green), Lef1 coupled to Alexa Fluor ® 594 (red), and DAPI (nuclei; blue) under a confocal microscope. Arrows point to nuclear colocalization. Scale bar = 100 and 20 μm inset images. b and c Expression profile of Tnfrsf11b and Ccn1/Cyr61 in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR, and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture value on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p

    Techniques Used: Expressing, Derivative Assay, Incubation, Microscopy, Quantitative RT-PCR

    Neurites cross the microgrooves to the axonal side of the microfluidic device Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. ( a and b ) The presence of neurites reaching MSCs was evaluated on day 7 of coculture by IF using an antibody directed against a neuronal specific marker (β-III Tubulin) coupled to Alexa Fluor ® 488 (green), and DAPI (nuclei; blue) under a confocal microscope. Actin filaments of MSCs were stained using Alexa Fluor ® 568 (red)-conjugated phalloidin. Arrows point to neurites. Scale bar = 100 µm a , 50 µm b
    Figure Legend Snippet: Neurites cross the microgrooves to the axonal side of the microfluidic device Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. ( a and b ) The presence of neurites reaching MSCs was evaluated on day 7 of coculture by IF using an antibody directed against a neuronal specific marker (β-III Tubulin) coupled to Alexa Fluor ® 488 (green), and DAPI (nuclei; blue) under a confocal microscope. Actin filaments of MSCs were stained using Alexa Fluor ® 568 (red)-conjugated phalloidin. Arrows point to neurites. Scale bar = 100 µm a , 50 µm b

    Techniques Used: Derivative Assay, Incubation, Marker, Microscopy, Staining

    DRG neurons enhance the osteoblast differentiation ability of MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a–d Expression profile of Runx2 , Sp7 , Col1a1 , and Bglap in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture levels on day 4. e and f Alp activity in MSCs was analyzed at 4 and 7 days of coculture by Alp activity quantification assay and cytochemical staining. Scale bar = 300 µm. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p
    Figure Legend Snippet: DRG neurons enhance the osteoblast differentiation ability of MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a–d Expression profile of Runx2 , Sp7 , Col1a1 , and Bglap in MSCs was assessed at 4 and 7 days of coculture by RT-qPCR and depicted as a relative ratio to the housekeeping gene Hprt1 normalized to the monoculture levels on day 4. e and f Alp activity in MSCs was analyzed at 4 and 7 days of coculture by Alp activity quantification assay and cytochemical staining. Scale bar = 300 µm. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. * p

    Techniques Used: Derivative Assay, Incubation, Expressing, Quantitative RT-PCR, ALP Assay, Activity Assay, Staining

    DRG neurons stimulate the metabolic activity in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a The DNA concentration of MSCs was determined at 4 and 7 days of coculture by CyQUANT™ Cell Proliferation Assay. b The relative metabolic activity of MSCs was measured at 4 and 7 days of coculture by resazurin-based assay and normalized to the monoculture levels on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. ** p
    Figure Legend Snippet: DRG neurons stimulate the metabolic activity in MSCs Sensory neurons derived from rat DRG (5 × 10 4 cells/cm 2 ) and rat bone marrow MSCs (10 4 cells/cm 2 ) were cocultured in microfluidic devices for 7 days. DRG neurons were maintained in DMEM supplemented with 2% (v/v) B-27 and 1 μM AraC; MSCs were incubated in OIM composed of DMEM-low glucose with 10% (v/v) FBS, 1 × 10 −9 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. a The DNA concentration of MSCs was determined at 4 and 7 days of coculture by CyQUANT™ Cell Proliferation Assay. b The relative metabolic activity of MSCs was measured at 4 and 7 days of coculture by resazurin-based assay and normalized to the monoculture levels on day 4. Data expressed as mean ± SD. ( n ) indicates the total number of samples for each group. ** p

    Techniques Used: Activity Assay, Derivative Assay, Incubation, Concentration Assay, CyQUANT Assay, Proliferation Assay, Resazurin Assay

    18) Product Images from "Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt\u2013\u03b2-catenin signaling"

    Article Title: Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt\u2013\u03b2-catenin signaling

    Journal: Blood

    doi: 10.1182/blood-2012-02-412890

    Microarray assay of gene expression in CML CD34 + cells cocultured with and without MSCs and with or without IM. (A) The number of differentially expressed genes when comparing the different treatments and (B) the interactions between MSCs and IM in determining
    Figure Legend Snippet: Microarray assay of gene expression in CML CD34 + cells cocultured with and without MSCs and with or without IM. (A) The number of differentially expressed genes when comparing the different treatments and (B) the interactions between MSCs and IM in determining

    Techniques Used: Microarray, Expressing

    MSCs protect CML LSCs capable of engrafting NSG mice from TKI treatment. CML CD34 + cells (2 × 10 6 cells per mouse) from 3 patients were cultured for 96 hours with and without MSCs and with or without IM and transplanted into NSG mice. Mice were
    Figure Legend Snippet: MSCs protect CML LSCs capable of engrafting NSG mice from TKI treatment. CML CD34 + cells (2 × 10 6 cells per mouse) from 3 patients were cultured for 96 hours with and without MSCs and with or without IM and transplanted into NSG mice. Mice were

    Techniques Used: Mouse Assay, Cell Culture

    Role of N-cadherin in MSC-mediated protection of CML stem/progenitor cells from TKI treatment. (A) CML CD34 + cells cocultured on MSCs without IM (left) and with IM (right) adhered to and migrated underneath (right, arrowhead) MSCs. (B) The fraction of
    Figure Legend Snippet: Role of N-cadherin in MSC-mediated protection of CML stem/progenitor cells from TKI treatment. (A) CML CD34 + cells cocultured on MSCs without IM (left) and with IM (right) adhered to and migrated underneath (right, arrowhead) MSCs. (B) The fraction of

    Techniques Used:

    Enhanced Wnt–β-catenin signaling in TKI-treated CML stem/progenitor cells cocultured with MSCs. (A) Western blotting for P-Crkl, N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34 + cells cultured with and without
    Figure Legend Snippet: Enhanced Wnt–β-catenin signaling in TKI-treated CML stem/progenitor cells cocultured with MSCs. (A) Western blotting for P-Crkl, N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34 + cells cultured with and without

    Techniques Used: Western Blot, Cell Culture

    MSCs protect CML CD34 + CD38 − and CD34 + CD38 + cells from TKI treatment. Primary CML and normal (NL) progenitor (CD34 + ) cells were stained with CFSE. CFSE + primitive cells (Lin − CD34 + CD38 − ) and committed cells (Lin − CD34 + CD38
    Figure Legend Snippet: MSCs protect CML CD34 + CD38 − and CD34 + CD38 + cells from TKI treatment. Primary CML and normal (NL) progenitor (CD34 + ) cells were stained with CFSE. CFSE + primitive cells (Lin − CD34 + CD38 − ) and committed cells (Lin − CD34 + CD38

    Techniques Used: Staining

    19) Product Images from "Overexpression of FABP3 Inhibits Human Bone Marrow Derived Mesenchymal Stem Cell Proliferation but Enhances their Survival in Hypoxia"

    Article Title: Overexpression of FABP3 Inhibits Human Bone Marrow Derived Mesenchymal Stem Cell Proliferation but Enhances their Survival in Hypoxia

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2014.02.015

    (A) Evidence of FABP3 overexpression in MSC FABP3LV (rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC FABP3LV , but not in vector control MSCs (red, a) and (C) MSC FABP3LV proteins were around the cellular nucleus under normoxic
    Figure Legend Snippet: (A) Evidence of FABP3 overexpression in MSC FABP3LV (rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC FABP3LV , but not in vector control MSCs (red, a) and (C) MSC FABP3LV proteins were around the cellular nucleus under normoxic

    Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    FABP3 expression pattern in MSCs. (A, a) FABP3 mRNA expression pattern in hypoxic human MSCs: Y axis: relative gene expression level, and X axis: cell group (n=12), * stands for p value, H vs. N: * p
    Figure Legend Snippet: FABP3 expression pattern in MSCs. (A, a) FABP3 mRNA expression pattern in hypoxic human MSCs: Y axis: relative gene expression level, and X axis: cell group (n=12), * stands for p value, H vs. N: * p

    Techniques Used: Expressing

    (A) Evidence of FABP3 overexpression in MSC FABP3LV (rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC FABP3LV , but not in vector control MSCs (red, a) and (C) MSC FABP3LV proteins were around the cellular nucleus under normoxic
    Figure Legend Snippet: (A) Evidence of FABP3 overexpression in MSC FABP3LV (rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC FABP3LV , but not in vector control MSCs (red, a) and (C) MSC FABP3LV proteins were around the cellular nucleus under normoxic

    Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    20) Product Images from "Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts"

    Article Title: Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S71466

    Effects of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on intratumoral microvessel density, cellular apoptosis and proliferation in gastric cancer xenografts in nude mice. Notes: The microvessel density was assessed using a monoclonal mouse anti-human CD31 antibody, cellular apoptosis was detected by TUNEL assay using an in situ apoptosis detection kit, and the proliferation was evaluated using immunostaining of PCNA with a monoclonal primary mouse anti-human PCNA antibody. ( A ) Representative tumor sections stained with an anti-CD31 antibody from different treatment groups, ( B ) bar graph to show the effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on intratumoral microvessel density in nude mice, nude mice treated with MSCs-NK4 had lowest intratumoral microvessel density among all treatment groups, ( C ) representative tumor sections stained with TUNEL where brown cells indicated by arrows are apoptotic cells, ( D ) bar graph to show the effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the apoptosis of tumor cells in vivo, nude mice treated with MSCs-NK4 had highest apoptotic index among all treatment groups, ( E ) representative tumor sections stained with anti-PCNA antibody, and ( F ) effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the proliferation of tumor cells in nude mice, nude mice treated with MSCs-NK4 had lowest proliferation index among all treatment groups. * P
    Figure Legend Snippet: Effects of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on intratumoral microvessel density, cellular apoptosis and proliferation in gastric cancer xenografts in nude mice. Notes: The microvessel density was assessed using a monoclonal mouse anti-human CD31 antibody, cellular apoptosis was detected by TUNEL assay using an in situ apoptosis detection kit, and the proliferation was evaluated using immunostaining of PCNA with a monoclonal primary mouse anti-human PCNA antibody. ( A ) Representative tumor sections stained with an anti-CD31 antibody from different treatment groups, ( B ) bar graph to show the effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on intratumoral microvessel density in nude mice, nude mice treated with MSCs-NK4 had lowest intratumoral microvessel density among all treatment groups, ( C ) representative tumor sections stained with TUNEL where brown cells indicated by arrows are apoptotic cells, ( D ) bar graph to show the effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the apoptosis of tumor cells in vivo, nude mice treated with MSCs-NK4 had highest apoptotic index among all treatment groups, ( E ) representative tumor sections stained with anti-PCNA antibody, and ( F ) effect of treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the proliferation of tumor cells in nude mice, nude mice treated with MSCs-NK4 had lowest proliferation index among all treatment groups. * P

    Techniques Used: Mouse Assay, TUNEL Assay, In Situ, Immunostaining, Staining, In Vivo

    Effects of MKN45 or GES-1 cell-conditioned medium on the migratory ability of MSCs and MSCs-NK4 toward gastric cancer cells determined using a Transwell migration assay. Notes: MNK45 or GES-1 cells were cultured for 24 hours in serum-free medium and then plated onto the bottom wells. MSCs, MSCs-NK4, or human fibroblasts cultured in serum-free medium were seeded onto the upper chamber and cultured for 24 hours. The MSCs, MSCs-NK4, or human fibroblasts that attached to the top side of the membrane were removed, and the cells that migrated onto the bottom side were fixed, stained, and counted (five fields per well) at 10× magnification using a microscope. ( A ) MKN45 cell-conditioned medium significantly stimulated the directional migration of MSCs and MSCs-NK4 compared to human fibroblasts. MSCs and MSCs-NK4 significantly migrated to MKN45 cell-conditioned medium, whereas GES-1 cell-conditioned medium or unconditioned medium did not promote the directional migration of MSCs or MSCs-NK4. *** P
    Figure Legend Snippet: Effects of MKN45 or GES-1 cell-conditioned medium on the migratory ability of MSCs and MSCs-NK4 toward gastric cancer cells determined using a Transwell migration assay. Notes: MNK45 or GES-1 cells were cultured for 24 hours in serum-free medium and then plated onto the bottom wells. MSCs, MSCs-NK4, or human fibroblasts cultured in serum-free medium were seeded onto the upper chamber and cultured for 24 hours. The MSCs, MSCs-NK4, or human fibroblasts that attached to the top side of the membrane were removed, and the cells that migrated onto the bottom side were fixed, stained, and counted (five fields per well) at 10× magnification using a microscope. ( A ) MKN45 cell-conditioned medium significantly stimulated the directional migration of MSCs and MSCs-NK4 compared to human fibroblasts. MSCs and MSCs-NK4 significantly migrated to MKN45 cell-conditioned medium, whereas GES-1 cell-conditioned medium or unconditioned medium did not promote the directional migration of MSCs or MSCs-NK4. *** P

    Techniques Used: Transwell Migration Assay, Cell Culture, Staining, Microscopy, Migration

    Characterization of MSCs-NK4 ( A ) and MSCs-GFP ( B ) phenotypes. Notes: After transduction of lentiviral vectors (Lenti-NK4 or Lenti-GFP), MSCs-NK4 and MSCs-GFP remained negative for CD45 and CD34 but positive for CD44 and CD105. Abbreviations: MSCs, mesenchymal stem cells; GFP, green fluorescent protein; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.
    Figure Legend Snippet: Characterization of MSCs-NK4 ( A ) and MSCs-GFP ( B ) phenotypes. Notes: After transduction of lentiviral vectors (Lenti-NK4 or Lenti-GFP), MSCs-NK4 and MSCs-GFP remained negative for CD45 and CD34 but positive for CD44 and CD105. Abbreviations: MSCs, mesenchymal stem cells; GFP, green fluorescent protein; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

    Techniques Used: Transduction

    Transduction of NK4 cDNA into human MSCs using a lentiviral vector and characterization. Notes: The recombinant pGC-FU-GFP-NK4-plasmids, the construction plasmids Helper1.0, and the envelope plasmids Helper2.0 (GeneChem Co., Ltd., Shanghai, People’s Republic of China) were first cotransfected into 293T cells. MSCs were then transduced with Lenti-NK4 (MSCs-NK4) or Lenti-GFP (MSCs-GFP) at varying multiplicity of infection (MOI). ( A ) Expression of GFP in MSCs observed at day three after Lenti-NK4 infection at MOI of 10, 20, 50, or 100 under fluorescence microscopy, ( B ) transduction efficiency of Lenti-NK4 in MSCs determined by flow cytometry with an GFP marker which was 87.8% of the enriched GFP-expressing MSC population upon sorting with an MOI of 50, ( C ) effect of transduction with Lenti-NK4 at different MOIs from 1 to 100 in MSCs on the production of NK4 in the culture medium, ( D ) effect of time on the production of NK4 in the culture medium after MSCs were transduced with Lenti-NK4 (MOI =50), Lenti-GFP (MOI =50), or not transduced, ( E ) MSCs-NK4 with increased GFP expression observed at different time points (MOI =50, from Day 1 to Day 3), and ( F ) Western blot analysis of NK4-GFP fusion protein with a molecular weight of 84 kDa. Recombinant human HGF (Mr =83 kDa) was used as a positive control. * P
    Figure Legend Snippet: Transduction of NK4 cDNA into human MSCs using a lentiviral vector and characterization. Notes: The recombinant pGC-FU-GFP-NK4-plasmids, the construction plasmids Helper1.0, and the envelope plasmids Helper2.0 (GeneChem Co., Ltd., Shanghai, People’s Republic of China) were first cotransfected into 293T cells. MSCs were then transduced with Lenti-NK4 (MSCs-NK4) or Lenti-GFP (MSCs-GFP) at varying multiplicity of infection (MOI). ( A ) Expression of GFP in MSCs observed at day three after Lenti-NK4 infection at MOI of 10, 20, 50, or 100 under fluorescence microscopy, ( B ) transduction efficiency of Lenti-NK4 in MSCs determined by flow cytometry with an GFP marker which was 87.8% of the enriched GFP-expressing MSC population upon sorting with an MOI of 50, ( C ) effect of transduction with Lenti-NK4 at different MOIs from 1 to 100 in MSCs on the production of NK4 in the culture medium, ( D ) effect of time on the production of NK4 in the culture medium after MSCs were transduced with Lenti-NK4 (MOI =50), Lenti-GFP (MOI =50), or not transduced, ( E ) MSCs-NK4 with increased GFP expression observed at different time points (MOI =50, from Day 1 to Day 3), and ( F ) Western blot analysis of NK4-GFP fusion protein with a molecular weight of 84 kDa. Recombinant human HGF (Mr =83 kDa) was used as a positive control. * P

    Techniques Used: Transduction, Plasmid Preparation, Recombinant, Pyrolysis Gas Chromatography, Infection, Expressing, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Marker, Western Blot, Molecular Weight, Positive Control

    Effects of systemic administration of PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the growth of gastric tumor xenografts over 28 days in BALB/C nude mice. Notes: Tumor-bearing BALB/C nude mice were injected with MSCs-GFP or MSCs-NK4 (6×10 5 cells in 0.2 mL PBS), Lenti-NK4 (3×10 7 viral particles), or PBS (0.2 mL) in the lateral tail vein for systemic administration at 1, 7, 14, and 21 days (eight animals per group). Three different diameters of each tumor were measured twice a week. Animals were sacrificed on the 28th day after injections, tumor xenografts were excised and weighed. ( A ) Representative tumor-bearing mice treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4, ( B ) the growth curve of tumor xenografts in nude mice. The mice injected with MSCs-NK4 exhibited obvious inhibition of tumor xenograft growth compared to other treatment groups. ( C ) Tumor volume at days 7, 14, 21, and 28 in mice injected with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tumor volume in mice treated with MSCs-NK4 was smallest among all groups. ( D ) Tumor weight in nude mice at day 28 after treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. Tumor burden in MSCs-NK4 treated mice was lower than mice treated with PBS or MSCs-GFP. ( E ) Histological score of necrosis in tumor xenografts in nude mice treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tumor necrosis scores in MSCs-NK4 treated mice were highest among all treatment groups. ( F ) H E staining of tumor tissues observed by light microscopy where tumor necrosis areas are indicated by arrows, and ( G ) presence of MSCs-NK4 in tumor tissues in vivo after nude mice were injected with MSCs-NK4 via the tail vein. Tumor xenografts were snap-frozen, cryosectioned and then examined under fluorescent microscope. * P
    Figure Legend Snippet: Effects of systemic administration of PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 on the growth of gastric tumor xenografts over 28 days in BALB/C nude mice. Notes: Tumor-bearing BALB/C nude mice were injected with MSCs-GFP or MSCs-NK4 (6×10 5 cells in 0.2 mL PBS), Lenti-NK4 (3×10 7 viral particles), or PBS (0.2 mL) in the lateral tail vein for systemic administration at 1, 7, 14, and 21 days (eight animals per group). Three different diameters of each tumor were measured twice a week. Animals were sacrificed on the 28th day after injections, tumor xenografts were excised and weighed. ( A ) Representative tumor-bearing mice treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4, ( B ) the growth curve of tumor xenografts in nude mice. The mice injected with MSCs-NK4 exhibited obvious inhibition of tumor xenograft growth compared to other treatment groups. ( C ) Tumor volume at days 7, 14, 21, and 28 in mice injected with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tumor volume in mice treated with MSCs-NK4 was smallest among all groups. ( D ) Tumor weight in nude mice at day 28 after treatment with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. Tumor burden in MSCs-NK4 treated mice was lower than mice treated with PBS or MSCs-GFP. ( E ) Histological score of necrosis in tumor xenografts in nude mice treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tumor necrosis scores in MSCs-NK4 treated mice were highest among all treatment groups. ( F ) H E staining of tumor tissues observed by light microscopy where tumor necrosis areas are indicated by arrows, and ( G ) presence of MSCs-NK4 in tumor tissues in vivo after nude mice were injected with MSCs-NK4 via the tail vein. Tumor xenografts were snap-frozen, cryosectioned and then examined under fluorescent microscope. * P

    Techniques Used: Mouse Assay, Injection, Inhibition, Staining, Light Microscopy, In Vivo, Microscopy

    21) Product Images from "Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis"

    Article Title: Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2018.0295

    Lentiviral dose response. (A) Luciferase activities, normalized by DNA content, of 4 week pellets are shown for MSCs transduced with variable doses of LV.COL2-GF or LV.CMV-GF. ( B, C ) Total GAG and DNA contents (μg/pellet) of additional pellets for the dose groups tested in (A) . Significance notations: δ, compared to 0 vp/cell; ϕ, compared to 625 vp/cell; ω, compared to 1250 vp/cell; θ, compared to 2500 vp/cell; *, compared to 625 vp/cell COL2-GF; ‡, compared to 625 vp/cell CMV-GF. (D) Toluidine Blue staining of sections from representative pellets for select test doses shown in (A–C) . Scale bars = 500 μm. GAG, glycosaminoglycan; vp, viral particles.
    Figure Legend Snippet: Lentiviral dose response. (A) Luciferase activities, normalized by DNA content, of 4 week pellets are shown for MSCs transduced with variable doses of LV.COL2-GF or LV.CMV-GF. ( B, C ) Total GAG and DNA contents (μg/pellet) of additional pellets for the dose groups tested in (A) . Significance notations: δ, compared to 0 vp/cell; ϕ, compared to 625 vp/cell; ω, compared to 1250 vp/cell; θ, compared to 2500 vp/cell; *, compared to 625 vp/cell COL2-GF; ‡, compared to 625 vp/cell CMV-GF. (D) Toluidine Blue staining of sections from representative pellets for select test doses shown in (A–C) . Scale bars = 500 μm. GAG, glycosaminoglycan; vp, viral particles.

    Techniques Used: Luciferase, Transduction, Staining

    22) Product Images from "Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells"

    Article Title: Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2015.0533

    To enhance EC attachment, ECs were cultured in EGM for 3 days before co-culture with MSCs or exposure to 10% cyclic tensile strain. From days 0 to 14, ECs, MSCs, and MSC-EC co-cultures were all cultured in OEM and either exposed to 10% cyclic tensile
    Figure Legend Snippet: To enhance EC attachment, ECs were cultured in EGM for 3 days before co-culture with MSCs or exposure to 10% cyclic tensile strain. From days 0 to 14, ECs, MSCs, and MSC-EC co-cultures were all cultured in OEM and either exposed to 10% cyclic tensile

    Techniques Used: Cell Culture, Co-Culture Assay

    Representative images demonstrating that ECs elongated in response to 10% cyclic tensile strain, while MSCs and MSC-EC co-cultures elongated and aligned perpendicular to the direction of strain. Scale bars, 100 μm (200 μm
    Figure Legend Snippet: Representative images demonstrating that ECs elongated in response to 10% cyclic tensile strain, while MSCs and MSC-EC co-cultures elongated and aligned perpendicular to the direction of strain. Scale bars, 100 μm (200 μm

    Techniques Used:

    Representative images demonstrating that ECs maintained expression of CD31 and VE-Cdh in co-cultures with MSCs and in response to 10% cyclic tensile strain, indicating that EC phenotypic stability was preserved. Scale bars, 100 μm. Color
    Figure Legend Snippet: Representative images demonstrating that ECs maintained expression of CD31 and VE-Cdh in co-cultures with MSCs and in response to 10% cyclic tensile strain, indicating that EC phenotypic stability was preserved. Scale bars, 100 μm. Color

    Techniques Used: Expressing

    23) Product Images from "Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome"

    Article Title: Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02491-7

    Exosomes from LPS-primed MSCs have a unique cell surface marker profile. Exosomes were isolated from BM-MSCs that were either primed with LPS (LPS-MSC exosomes) or without LPS (MSC exosomes) for 24 h. The isolated exosomes were stained overnight with 37 different bead surface marker populations and compared by mean fluorescence intensity. Results are from two replicates of two independent studies. Groups were compared by Kruskal-Wallis with a Dunn post-test. N = 4, * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005
    Figure Legend Snippet: Exosomes from LPS-primed MSCs have a unique cell surface marker profile. Exosomes were isolated from BM-MSCs that were either primed with LPS (LPS-MSC exosomes) or without LPS (MSC exosomes) for 24 h. The isolated exosomes were stained overnight with 37 different bead surface marker populations and compared by mean fluorescence intensity. Results are from two replicates of two independent studies. Groups were compared by Kruskal-Wallis with a Dunn post-test. N = 4, * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005

    Techniques Used: Marker, Isolation, Staining, Fluorescence

    24) Product Images from "PDGF-AA mediates mesenchymal stromal cell chemotaxis to the head and neck squamous cell carcinoma tumor microenvironment"

    Article Title: PDGF-AA mediates mesenchymal stromal cell chemotaxis to the head and neck squamous cell carcinoma tumor microenvironment

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-1091-6

    Seven chemokines and growth factors were noted to be highly secreted in the conditioned media of JHU-011 and JHU-019. Of those, only recombinant IL-6 and PDGF isoforms AA, BB, and AB independently caused significant migration of MSCs to levels comparable to OPSCC cell line JHU-019 when compared to 1% BSA treated media ( a ). Neutralizing antibodies to all PDGF isoforms ( b ) and IL-6 ( c ) in the conditioned media of cell lines JHU-011, -012, and -019 and/or -022 resulted in a significant reduction in MSC migration. PDGFRα and PDGFRβ was expressed on MSCs and not JHU-011, -012, -019 or JHU-022. 3T3 cells were used as a negative control ( d ). Only blockade of PDGFRα but not PDGFRβ resulted in a significant reduction in JHU-011 induced MSC chemotaxis. Two MSC passes P3 and P5 were used to ensure MSC passage did not affect PDGFR expression. Each experiment was performed in triplicate for n ≥ 3 and unless where indicated (*p
    Figure Legend Snippet: Seven chemokines and growth factors were noted to be highly secreted in the conditioned media of JHU-011 and JHU-019. Of those, only recombinant IL-6 and PDGF isoforms AA, BB, and AB independently caused significant migration of MSCs to levels comparable to OPSCC cell line JHU-019 when compared to 1% BSA treated media ( a ). Neutralizing antibodies to all PDGF isoforms ( b ) and IL-6 ( c ) in the conditioned media of cell lines JHU-011, -012, and -019 and/or -022 resulted in a significant reduction in MSC migration. PDGFRα and PDGFRβ was expressed on MSCs and not JHU-011, -012, -019 or JHU-022. 3T3 cells were used as a negative control ( d ). Only blockade of PDGFRα but not PDGFRβ resulted in a significant reduction in JHU-011 induced MSC chemotaxis. Two MSC passes P3 and P5 were used to ensure MSC passage did not affect PDGFR expression. Each experiment was performed in triplicate for n ≥ 3 and unless where indicated (*p

    Techniques Used: Recombinant, Migration, Negative Control, Chemotaxis Assay, Expressing

    MSCs reside in the OPSCC (human tonsil) tumor stromal microenvironment. The black arrows denote the tumor microenvironment (TM). Representative immunohistological sections of human OPSCC demonstrates the presence of a distinct population of gremlin-1+ MSCs in the TM ( a – c ). The black square denotes areas viewed at higher magnification. Cells positive for the myofibroblast/CAF marker, α-SMA+ , were also present within the TM ( d – f ). A small population of CD14+ lymphocytes are seen at higher power magnification, representing cells of hematopoietic lineage ( g – i ). The above photomicrographs are serial sections (4 μm thickness)
    Figure Legend Snippet: MSCs reside in the OPSCC (human tonsil) tumor stromal microenvironment. The black arrows denote the tumor microenvironment (TM). Representative immunohistological sections of human OPSCC demonstrates the presence of a distinct population of gremlin-1+ MSCs in the TM ( a – c ). The black square denotes areas viewed at higher magnification. Cells positive for the myofibroblast/CAF marker, α-SMA+ , were also present within the TM ( d – f ). A small population of CD14+ lymphocytes are seen at higher power magnification, representing cells of hematopoietic lineage ( g – i ). The above photomicrographs are serial sections (4 μm thickness)

    Techniques Used: Marker

    Conditioned media from 3 well characterized OPSCC cells lines (JHU-011, -012, and -019) caused significant migration and invasion of MSCs. Following 24-h incubation with conditioned media from JHU-011, -012, and -019, a > 60% increase in MSC migration was observed when compared to OKT controls ( a ). MSCs were also observed to have a significant increased capacity for invasion > 50% compared to that caused by the conditioned media from OKT controls ( b ). All data are expressed as a percentage increase in MSC migration and invasion normalized to OKT controls. Each experiment was performed in triplicate for n ≥ 3 (p
    Figure Legend Snippet: Conditioned media from 3 well characterized OPSCC cells lines (JHU-011, -012, and -019) caused significant migration and invasion of MSCs. Following 24-h incubation with conditioned media from JHU-011, -012, and -019, a > 60% increase in MSC migration was observed when compared to OKT controls ( a ). MSCs were also observed to have a significant increased capacity for invasion > 50% compared to that caused by the conditioned media from OKT controls ( b ). All data are expressed as a percentage increase in MSC migration and invasion normalized to OKT controls. Each experiment was performed in triplicate for n ≥ 3 (p

    Techniques Used: Migration, Incubation

    25) Product Images from "Adenosine from a biologic source regulates neutrophil extracellular traps (NETs)"

    Article Title: Adenosine from a biologic source regulates neutrophil extracellular traps (NETs)

    Journal: Journal of leukocyte biology

    doi: 10.1002/JLB.3VMA0918-374R

    Mesenchymal stromal cells (MSCs) suppress neutrophil extracellular trap (NET) formation in vitro. Human neutrophils were cocultured with MSCs for 30 min prior to stimulation with PMA. SYTOX green fluorescence staining ( A ) shows increase extracellular DNA, 2 h after PMA administration, and a reduction in cells cocultured with MSCs. MSCs alone without neutrophils showed no fluorescence (not shown). Quantification of green fluorescence ( B ; n = 18) as well as HNE-DNA ELISA assay ( C ) showed strong NET-inhibitory effect of MSCs ( n = 13). Data are expressed as mean ± SD, *** P
    Figure Legend Snippet: Mesenchymal stromal cells (MSCs) suppress neutrophil extracellular trap (NET) formation in vitro. Human neutrophils were cocultured with MSCs for 30 min prior to stimulation with PMA. SYTOX green fluorescence staining ( A ) shows increase extracellular DNA, 2 h after PMA administration, and a reduction in cells cocultured with MSCs. MSCs alone without neutrophils showed no fluorescence (not shown). Quantification of green fluorescence ( B ; n = 18) as well as HNE-DNA ELISA assay ( C ) showed strong NET-inhibitory effect of MSCs ( n = 13). Data are expressed as mean ± SD, *** P

    Techniques Used: In Vitro, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay

    26) Product Images from "Combined therapy with adipose tissue-derived mesenchymal stromal cells and meglumine antimoniate controls lesion development and parasite load in murine cutaneous leishmaniasis caused by Leishmania amazonensis"

    Article Title: Combined therapy with adipose tissue-derived mesenchymal stromal cells and meglumine antimoniate controls lesion development and parasite load in murine cutaneous leishmaniasis caused by Leishmania amazonensis

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-01889-z

    Wound healing scratch assay. 3T3 cells were cultured until reaching 80% confluence. The cell monolayers were scratched, and the medium of the culture was changed as follows: RPMI (negative control—Supp, Fig. 1 ); DMEM + FBS (positive control—Supp, Fig. 1 ); RPMI + supernatant of infected macrophages (Mɸ + La); RPMI + supernatant of infected macrophages cultured with conditioned medium of MSCs (Mɸ + La + AD-MSC SD and Mɸ + La + BM-MSC SD). The scratch on the cell monolayer was photographed every 20 min for 96 h and analyzed by IncuCyte ZOOM 2015A. Values show the mean ± standard deviation
    Figure Legend Snippet: Wound healing scratch assay. 3T3 cells were cultured until reaching 80% confluence. The cell monolayers were scratched, and the medium of the culture was changed as follows: RPMI (negative control—Supp, Fig. 1 ); DMEM + FBS (positive control—Supp, Fig. 1 ); RPMI + supernatant of infected macrophages (Mɸ + La); RPMI + supernatant of infected macrophages cultured with conditioned medium of MSCs (Mɸ + La + AD-MSC SD and Mɸ + La + BM-MSC SD). The scratch on the cell monolayer was photographed every 20 min for 96 h and analyzed by IncuCyte ZOOM 2015A. Values show the mean ± standard deviation

    Techniques Used: Wound Healing Assay, Cell Culture, Negative Control, Positive Control, Infection, Standard Deviation

    27) Product Images from "Toxicity of JQ1 in neuronal derivatives of human umbilical cord mesenchymal stem cells"

    Article Title: Toxicity of JQ1 in neuronal derivatives of human umbilical cord mesenchymal stem cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26127

    Effect of JQ1 on expression of neural markers MSCs and NDs were untreated (−) or treated (+) with JQ1 for 48 hours. ( A and B ) Immunocytochemical analysis of expression of neural proteins TUJ1, Nestin, and NeuN, in MSCs and NDs in the absence or presence of JQ1, respectively. Scale bars represent 50 μm (Magnification: 10X) and 20 μm in high magnification merged inserts (Magnification: 40X), respectively. ( C ) Quantification of normalized fluorescent intensities of neural proteins in MSCs and NDs treated with and without JQ1 using ImageJ software. ( D ) Transcriptional analysis of neural genes, TUJ1 , Nestin , and PAX6 as determined by qRT-PCR. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of JQ1 on expression of neural markers MSCs and NDs were untreated (−) or treated (+) with JQ1 for 48 hours. ( A and B ) Immunocytochemical analysis of expression of neural proteins TUJ1, Nestin, and NeuN, in MSCs and NDs in the absence or presence of JQ1, respectively. Scale bars represent 50 μm (Magnification: 10X) and 20 μm in high magnification merged inserts (Magnification: 40X), respectively. ( C ) Quantification of normalized fluorescent intensities of neural proteins in MSCs and NDs treated with and without JQ1 using ImageJ software. ( D ) Transcriptional analysis of neural genes, TUJ1 , Nestin , and PAX6 as determined by qRT-PCR. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p

    Techniques Used: Expressing, Software, Quantitative RT-PCR

    Effect of JQ1 on morphology, viability, and growth of MSCs and derivatives MSCs were cultured in culture medium (CM) or differentiation media for induction into adipogenic, chondrogenic, osteogenic, and neuronal derivatives (ADs, CDs, ODs, and NDs, respectively). ( A ) Cell morphology was visualized by phase contrast microscopy. Scale bars represent 100 µm (Magnification: 4X). Arrows in JQ1 treated NDs point to rounded cells. ( B ) Relative growth of MSCs and their derivatives in the absence or presence of JQ1. ( C ) Graphical representation of the percentage of viable cells as determined by trypan blue staining. ( D ) Representative flow cytometric analysis of MSCs and NDs to determine the MSC specific markers, CD90, CD73, CD44, and CD105. ( E ) Graphical representation of flow cytometric data showing percentage of cells positive for MSC markers. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of JQ1 on morphology, viability, and growth of MSCs and derivatives MSCs were cultured in culture medium (CM) or differentiation media for induction into adipogenic, chondrogenic, osteogenic, and neuronal derivatives (ADs, CDs, ODs, and NDs, respectively). ( A ) Cell morphology was visualized by phase contrast microscopy. Scale bars represent 100 µm (Magnification: 4X). Arrows in JQ1 treated NDs point to rounded cells. ( B ) Relative growth of MSCs and their derivatives in the absence or presence of JQ1. ( C ) Graphical representation of the percentage of viable cells as determined by trypan blue staining. ( D ) Representative flow cytometric analysis of MSCs and NDs to determine the MSC specific markers, CD90, CD73, CD44, and CD105. ( E ) Graphical representation of flow cytometric data showing percentage of cells positive for MSC markers. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p

    Techniques Used: Cell Culture, Microscopy, Staining, Flow Cytometry

    Effect of JQ1 on the expression of Caspase 9 and Cytochrome C MSCs and NDs untreated (−) and treated (+) with JQ1 for 48 hours and subjected to analysis. ( A ) Representative flow cytomeric plots of cells stained with Annexin-V/FITC and PI. ( B ) Graphical representation of the average percentage of dead cells as determined by flow cytometry, error bars represent SEM of three independent experiments ( n = 3). ( C ) Immunocytochemical analysis of Caspase 9 showing protein expression in NDs treated with JQ1. Scale bars represent 50 μm (Magnification: 10X) and 20 μm in high magnification merged insert (Magnification: 40X), respectively. ( D ) Quantification of normalized fluorescent intensity of Caspase 9 expression in NDs using ImageJ software. * p
    Figure Legend Snippet: Effect of JQ1 on the expression of Caspase 9 and Cytochrome C MSCs and NDs untreated (−) and treated (+) with JQ1 for 48 hours and subjected to analysis. ( A ) Representative flow cytomeric plots of cells stained with Annexin-V/FITC and PI. ( B ) Graphical representation of the average percentage of dead cells as determined by flow cytometry, error bars represent SEM of three independent experiments ( n = 3). ( C ) Immunocytochemical analysis of Caspase 9 showing protein expression in NDs treated with JQ1. Scale bars represent 50 μm (Magnification: 10X) and 20 μm in high magnification merged insert (Magnification: 40X), respectively. ( D ) Quantification of normalized fluorescent intensity of Caspase 9 expression in NDs using ImageJ software. * p

    Techniques Used: Expressing, Flow Cytometry, Staining, Cytometry, Software

    Proposed mechanism of action of JQ1 (A ) Transcriptional analysis of BRD4 , c - MYC , p53 , p21 , PUMA , NOXA , and BAX in MSCs and NDs untreated (−) and treated (+) with JQ1 as determined by qRT-PCR. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Proposed mechanism of action of JQ1 (A ) Transcriptional analysis of BRD4 , c - MYC , p53 , p21 , PUMA , NOXA , and BAX in MSCs and NDs untreated (−) and treated (+) with JQ1 as determined by qRT-PCR. Experiments were performed in triplicate and error bars represent SEM of three independent experiments ( n = 3). * p

    Techniques Used: Quantitative RT-PCR

    28) Product Images from "Use of a Combination Strategy to Improve Morphological and Functional Recovery in Rats With Chronic Spinal Cord Injury"

    Article Title: Use of a Combination Strategy to Improve Morphological and Functional Recovery in Rats With Chronic Spinal Cord Injury

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2020.00189

    Representative microphotographs of BrdU+/DCX+ cells at ventral horns of SCI rats after therapeutic intervention. In the first section, pkH26 positive cells (red), BrdU+ cells (green), Dcx+ cells (blue). Double-label (BrdU+/Dcx+; cyan), triple-labeling (BrdU+/Dcx+/pkH26+; yellow) show merged final section. (A) PBS-I, (B) SR + FGM-MSCs, (C) SR + INDP + FGM-MSCs, and (D) INDP. An asterisk (*) indicates neuroblasts with triple labeling. Arrows depict BrdU+/DCX+ cells. A higher number of neuroblasts was observed in the group with INDP. This is one representative photograph of three experiments. Scale bar 20 μm.
    Figure Legend Snippet: Representative microphotographs of BrdU+/DCX+ cells at ventral horns of SCI rats after therapeutic intervention. In the first section, pkH26 positive cells (red), BrdU+ cells (green), Dcx+ cells (blue). Double-label (BrdU+/Dcx+; cyan), triple-labeling (BrdU+/Dcx+/pkH26+; yellow) show merged final section. (A) PBS-I, (B) SR + FGM-MSCs, (C) SR + INDP + FGM-MSCs, and (D) INDP. An asterisk (*) indicates neuroblasts with triple labeling. Arrows depict BrdU+/DCX+ cells. A higher number of neuroblasts was observed in the group with INDP. This is one representative photograph of three experiments. Scale bar 20 μm.

    Techniques Used: Labeling

    29) Product Images from "Photoreceptor protection by mesenchymal stem cell transplantation identifies exosomal MiR-21 as a therapeutic for retinal degeneration"

    Article Title: Photoreceptor protection by mesenchymal stem cell transplantation identifies exosomal MiR-21 as a therapeutic for retinal degeneration

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-020-00636-4

    miR-21 deficiency aggravates N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and retinal degeneration, which is restrained by transplantation of mesenchymal stem cell (MSC)-derived exosomes. A Tracing of CD63-EGFP plasmid-transfected MSCs (green, CD63) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. MSCT mesenchymal stem cell transplantation, INL inner nuclear layer, ONL outer nuclear layer. Scale bar = 50 μm. B Tracing of PKH26-labeled exosomes (red) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. EXOT exosomal transplantation, NC negative control, injection of EXO without staining. Scale bar = 50 μm. C Tracing of PKH26-labeled, MSC-derived exosomes (red) in 661W cone photoreceptor cells during in vitro treatment. 661W cells are demonstrated by β-tubulin immunostaining for microtubules (green), counterstained by Hoechst 33342 (blue). NC negative control, treatment of EXO without staining. Scale bar = 10 μm. D Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of expression levels of multiple microRNAs in retina tissues, normalized to Rnu6 . Ctrl control. * P
    Figure Legend Snippet: miR-21 deficiency aggravates N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and retinal degeneration, which is restrained by transplantation of mesenchymal stem cell (MSC)-derived exosomes. A Tracing of CD63-EGFP plasmid-transfected MSCs (green, CD63) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. MSCT mesenchymal stem cell transplantation, INL inner nuclear layer, ONL outer nuclear layer. Scale bar = 50 μm. B Tracing of PKH26-labeled exosomes (red) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. EXOT exosomal transplantation, NC negative control, injection of EXO without staining. Scale bar = 50 μm. C Tracing of PKH26-labeled, MSC-derived exosomes (red) in 661W cone photoreceptor cells during in vitro treatment. 661W cells are demonstrated by β-tubulin immunostaining for microtubules (green), counterstained by Hoechst 33342 (blue). NC negative control, treatment of EXO without staining. Scale bar = 10 μm. D Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of expression levels of multiple microRNAs in retina tissues, normalized to Rnu6 . Ctrl control. * P

    Techniques Used: Transplantation Assay, Derivative Assay, Plasmid Preparation, Transfection, Injection, Labeling, Negative Control, Staining, In Vitro, Immunostaining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Exosomal miR-21 counteracts N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and retinal degeneration. Representative hematoxylin and eosin (H E) staining images of retinal tissues ( A ) and the corresponding quantitative analysis of outer nuclear layer (ONL) thickness ( B ). WT-EXOT transplantation of exosomes derived from wild-type mesenchymal stem cells (MSCs) after MNU injection, miR-21 −/− -EXOT transplantation of exosomes derived from miR-21-deficient MSCs after MNU injection, GCL ganglion cell layer, INL inner nuclear layer, ONH optic nerve head. Scale bars = 50 μm. * P
    Figure Legend Snippet: Exosomal miR-21 counteracts N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and retinal degeneration. Representative hematoxylin and eosin (H E) staining images of retinal tissues ( A ) and the corresponding quantitative analysis of outer nuclear layer (ONL) thickness ( B ). WT-EXOT transplantation of exosomes derived from wild-type mesenchymal stem cells (MSCs) after MNU injection, miR-21 −/− -EXOT transplantation of exosomes derived from miR-21-deficient MSCs after MNU injection, GCL ganglion cell layer, INL inner nuclear layer, ONH optic nerve head. Scale bars = 50 μm. * P

    Techniques Used: Staining, Transplantation Assay, Derivative Assay, Injection

    Intravitreal transplantation of mesenchymal stem cells (MSCs) counteracts N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and alleviates retinal degeneration. A Schematic diagram demonstrating the study design of in vivo experiments on MNU-induced retinal degeneration. B Biodistribution of PKH26-labeled MSCs in the eye after intravitreal injection for 6 h. NC negative control, injection of MSCs without staining. Scale bar = 2.5 mm. C Tracing of PKH26-labeled MSCs (red) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. MSCT mesenchymal stem cell transplantation after MNU injection, INL inner nuclear layer, ONL outer nuclear layer. Scale bar = 50 μm. Representative hematoxylin and eosin (H E) staining images of retinal tissues ( D ) and the corresponding quantitative analysis of ONL thickness ( E ). Ctrl control, no MNU treatment, GCL ganglion cell layer, ONH optic nerve head. Scale bars = 50 μm. * P
    Figure Legend Snippet: Intravitreal transplantation of mesenchymal stem cells (MSCs) counteracts N -methyl- N -nitrosourea (MNU)-induced photoreceptor apoptosis and alleviates retinal degeneration. A Schematic diagram demonstrating the study design of in vivo experiments on MNU-induced retinal degeneration. B Biodistribution of PKH26-labeled MSCs in the eye after intravitreal injection for 6 h. NC negative control, injection of MSCs without staining. Scale bar = 2.5 mm. C Tracing of PKH26-labeled MSCs (red) in the retina tissue counterstained by Hoechst 33342 (blue) after intravitreal injection for 24 h. MSCT mesenchymal stem cell transplantation after MNU injection, INL inner nuclear layer, ONL outer nuclear layer. Scale bar = 50 μm. Representative hematoxylin and eosin (H E) staining images of retinal tissues ( D ) and the corresponding quantitative analysis of ONL thickness ( E ). Ctrl control, no MNU treatment, GCL ganglion cell layer, ONH optic nerve head. Scale bars = 50 μm. * P

    Techniques Used: Transplantation Assay, In Vivo, Labeling, Injection, Negative Control, Staining

    30) Product Images from "Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study"

    Article Title: Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study

    Journal: Cytotechnology

    doi: 10.1007/s10616-014-9718-z

    Bone marrox ( BM ) MSCs reached 70 % confluency in a shorter period (12 days) when compared to WJ (15 days), AT (14 days) and UCB (23 days) derived MSCs
    Figure Legend Snippet: Bone marrox ( BM ) MSCs reached 70 % confluency in a shorter period (12 days) when compared to WJ (15 days), AT (14 days) and UCB (23 days) derived MSCs

    Techniques Used: Derivative Assay

    31) Product Images from "Interferon-? Regulates the Proliferation and Differentiation of Mesenchymal Stem Cells via Activation of Indoleamine 2,3 Dioxygenase (IDO)"

    Article Title: Interferon-? Regulates the Proliferation and Differentiation of Mesenchymal Stem Cells via Activation of Indoleamine 2,3 Dioxygenase (IDO)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014698

    IFN-γ inhibits the proliferation of mouse and human MSCs. A and B . Cell growth of human and mouse MSCs showing cumulative population doublings as a function of time in culture. Between P4 and P12, cells were cultured in the continuous presence of IFN-γ (100 IU/ml) and/or IDO inhibitors norharmane, D-1-methyl-tryptophan and L-1-methyl-tryptophan for 80 and 50 days respectively. At every passage, population doubling was calculated by the formula logN/log2 as described by Stenderup [63] where N is the ratio between the number of viable cells reaching confluence and the number of cells initially plated. Medium was changed every three days using α-MEM containing 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 20% or 10% non-inactivated FBS, for human and mouse cells respectively, specially tested for the ability to sustain the growth of MSCs. C. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without tryptophan and treated for 5 days with increasing concentrations of FBS (0, 0.1, 0.5, 2 or 10%) and IFN-γ (0, 1 or 10 IU/ml) D. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without serum and treated for 5 days with increasing concentrations of tryptophan (0, 0.1, 1, 5, 10 or 44.2 µM) and IFN-γ (0, 1 or 10 IU/ml) E. Number of viable mouse MSCs (passage 14) cultured in serum free DMEM F12 medium in the presence of 10 IU/ml IFN-γ, increasing concentrations of tryptophan (0, 0.1, 1, 5, and 10 µM) and/or IDO inhibitors D-1-methyl-tryptophan and L-1-methyl-tryptophan (100 µM) for 5 days. Mouse MSCs were cultured with 10% FBS as positive controls. F. Expression of full IDO1 mRNA in mouse MSCs (passage 20) as measured by qRT-PCR. Cells were grown in the presence of increasing concentrations of tryptophan (0, 1, 5, 10 and 44 µM) and/or IFN-γ (0, 2, 10 and 100 IU/ml) for 24 hours. Mouse MSCs were cultured with 10% FBS as positive controls. Data are mean ± standard error (SEM). *p
    Figure Legend Snippet: IFN-γ inhibits the proliferation of mouse and human MSCs. A and B . Cell growth of human and mouse MSCs showing cumulative population doublings as a function of time in culture. Between P4 and P12, cells were cultured in the continuous presence of IFN-γ (100 IU/ml) and/or IDO inhibitors norharmane, D-1-methyl-tryptophan and L-1-methyl-tryptophan for 80 and 50 days respectively. At every passage, population doubling was calculated by the formula logN/log2 as described by Stenderup [63] where N is the ratio between the number of viable cells reaching confluence and the number of cells initially plated. Medium was changed every three days using α-MEM containing 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 20% or 10% non-inactivated FBS, for human and mouse cells respectively, specially tested for the ability to sustain the growth of MSCs. C. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without tryptophan and treated for 5 days with increasing concentrations of FBS (0, 0.1, 0.5, 2 or 10%) and IFN-γ (0, 1 or 10 IU/ml) D. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without serum and treated for 5 days with increasing concentrations of tryptophan (0, 0.1, 1, 5, 10 or 44.2 µM) and IFN-γ (0, 1 or 10 IU/ml) E. Number of viable mouse MSCs (passage 14) cultured in serum free DMEM F12 medium in the presence of 10 IU/ml IFN-γ, increasing concentrations of tryptophan (0, 0.1, 1, 5, and 10 µM) and/or IDO inhibitors D-1-methyl-tryptophan and L-1-methyl-tryptophan (100 µM) for 5 days. Mouse MSCs were cultured with 10% FBS as positive controls. F. Expression of full IDO1 mRNA in mouse MSCs (passage 20) as measured by qRT-PCR. Cells were grown in the presence of increasing concentrations of tryptophan (0, 1, 5, 10 and 44 µM) and/or IFN-γ (0, 2, 10 and 100 IU/ml) for 24 hours. Mouse MSCs were cultured with 10% FBS as positive controls. Data are mean ± standard error (SEM). *p

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR

    32) Product Images from "Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions"

    Article Title: Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-1057-8

    Expression of CD39 and CD73 in NCx-MSCs and CeCa-MSCs. The expression of CD39 and CD73 ectonucleotidases was determined in NCx-MSC (n = 5) and CeCa-MSC (n = 5) cell membranes by flow cytometry analysis ( a ) and by immunocytochemical analysis ( b , c ) as described in “ Methods ” section. The arrows indicate typical cells stained with human anti-CD39 and anti-CD73 mAbs. The mean fluorescence intensity (MFI) ± SEM of 10,000 events ( a ), and the total expression density (TED) evaluated by digital pathology using the Aperio CS system ( c ) are shown. Secondary antibody alone was included as control (Ctl) for the experiments. The images were taken at ×20 magnification ( Scale bar 100 µm). Asterisk indicates significant differences (P
    Figure Legend Snippet: Expression of CD39 and CD73 in NCx-MSCs and CeCa-MSCs. The expression of CD39 and CD73 ectonucleotidases was determined in NCx-MSC (n = 5) and CeCa-MSC (n = 5) cell membranes by flow cytometry analysis ( a ) and by immunocytochemical analysis ( b , c ) as described in “ Methods ” section. The arrows indicate typical cells stained with human anti-CD39 and anti-CD73 mAbs. The mean fluorescence intensity (MFI) ± SEM of 10,000 events ( a ), and the total expression density (TED) evaluated by digital pathology using the Aperio CS system ( c ) are shown. Secondary antibody alone was included as control (Ctl) for the experiments. The images were taken at ×20 magnification ( Scale bar 100 µm). Asterisk indicates significant differences (P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence, CTL Assay

    Hydrolytic activity of CD39 and CD73 ectonucleotidases expressed in MSCs. A total of 1 × 10 5 CEMs derived from NCx-MSCs (n = 5) and CeCa-MSCs (n = 5) were cultured at 37 °C with 5 mM adenine nucleotides (ATP, ADP or AMP) in the presence or absence of POM-1 (specific CD39 inhibitor) or APCP (specific CD73 inhibitor). a Adenosine produced by the hydrolysis of nucleotides was analyzed by thin layer chromatography (TLC). The ATP, ADP and AMP hydrolysis products (marked with arrows ) at the end of MSC culture with different nucleotides are shown. ATP, ADP, AMP, inosine (Ino) and synthetic adenosine were used as markers. b Adenosine contained in supernatant samples was quantified every 60 min by ultra-performance liquid chromatography (UPLC), using standard concentrations of synthetic Ado ( upper ). A representative linear range between concentration and histogram integral area for Ado is shown ( lower ). c The concentration of Ado produced by the hydrolysis of ATP ( upper ), ADP ( middle ) and AMP ( lower ) during the period of MSC culture with the respective nucleotides is shown. Asterisk indicates significant (P
    Figure Legend Snippet: Hydrolytic activity of CD39 and CD73 ectonucleotidases expressed in MSCs. A total of 1 × 10 5 CEMs derived from NCx-MSCs (n = 5) and CeCa-MSCs (n = 5) were cultured at 37 °C with 5 mM adenine nucleotides (ATP, ADP or AMP) in the presence or absence of POM-1 (specific CD39 inhibitor) or APCP (specific CD73 inhibitor). a Adenosine produced by the hydrolysis of nucleotides was analyzed by thin layer chromatography (TLC). The ATP, ADP and AMP hydrolysis products (marked with arrows ) at the end of MSC culture with different nucleotides are shown. ATP, ADP, AMP, inosine (Ino) and synthetic adenosine were used as markers. b Adenosine contained in supernatant samples was quantified every 60 min by ultra-performance liquid chromatography (UPLC), using standard concentrations of synthetic Ado ( upper ). A representative linear range between concentration and histogram integral area for Ado is shown ( lower ). c The concentration of Ado produced by the hydrolysis of ATP ( upper ), ADP ( middle ) and AMP ( lower ) during the period of MSC culture with the respective nucleotides is shown. Asterisk indicates significant (P

    Techniques Used: Activity Assay, Derivative Assay, Cell Culture, Produced, Thin Layer Chromatography, Liquid Chromatography, Concentration Assay

    33) Product Images from "Human adult stem cells derived from adipose tissue and bone marrow attenuate enteric neuropathy in the guinea-pig model of acute colitis"

    Article Title: Human adult stem cells derived from adipose tissue and bone marrow attenuate enteric neuropathy in the guinea-pig model of acute colitis

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0231-x

    Phenotypic and functional validation of BM-MSCs and AT-MSCs. a BM-MSCs and AT-MSCs analysed for cell surface antigen expression of known positive (CD29, CD44, CD73, and CD90) and negative (CD34 and CD45) MSC markers. Red closed histograms represent MSCs labelled with antibodies against the surface antigen indicated on the right hand side of each row. Blue open histograms show isotype controls. BM-MSCs b and AT-MSCs b ′ adhered to plastic with a perceptible appearance typical of MSCs in culture. Scale bar = 200 μm. BM-MSCs and AT-MSCs cultured without c - d and with c ′- d ′ adipogenesis differentiation medium for 14 days and stained with Oil red O. Scale bar = 50 μm. BM-MSCs and AT-MSCs cultured without e - f and with e ′- f ′ osteogenesis differentiation medium for 21 days and stained with Alizarin red S. Scale bar = 200 μm. BM-MSCs bone marrow-derived mesenchymal stem cells, AT-BMCs adipose tissue-derived mesenchymal stem cells
    Figure Legend Snippet: Phenotypic and functional validation of BM-MSCs and AT-MSCs. a BM-MSCs and AT-MSCs analysed for cell surface antigen expression of known positive (CD29, CD44, CD73, and CD90) and negative (CD34 and CD45) MSC markers. Red closed histograms represent MSCs labelled with antibodies against the surface antigen indicated on the right hand side of each row. Blue open histograms show isotype controls. BM-MSCs b and AT-MSCs b ′ adhered to plastic with a perceptible appearance typical of MSCs in culture. Scale bar = 200 μm. BM-MSCs and AT-MSCs cultured without c - d and with c ′- d ′ adipogenesis differentiation medium for 14 days and stained with Oil red O. Scale bar = 50 μm. BM-MSCs and AT-MSCs cultured without e - f and with e ′- f ′ osteogenesis differentiation medium for 21 days and stained with Alizarin red S. Scale bar = 200 μm. BM-MSCs bone marrow-derived mesenchymal stem cells, AT-BMCs adipose tissue-derived mesenchymal stem cells

    Techniques Used: Functional Assay, Expressing, Cell Culture, Staining, Derivative Assay

    34) Product Images from "Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing"

    Article Title: Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0805-5

    Impact of radial shockwaves on multidifferentiation of MSCs. a Multidifferentiation tests of MSCs showed radial shockwave stimulation increased ALP activity but decreased formation of Oil Red-O-positive lipid droplets, indicating that shockwaves promote osteogenesis induction and block adipogenesis at the same time. No significant differences in the two cohorts of cells after chondrogenic induction. b Impact of shockwaves on MSC differentiation also reflected by mRNA levels of Runx-2 , Osterix , CEBP/α , PPARγ , Sox-9 , and Col-II . Quantitative PCR assay performed at least three times independently; representative result shown. *Statistically significant difference compared with control groups, P
    Figure Legend Snippet: Impact of radial shockwaves on multidifferentiation of MSCs. a Multidifferentiation tests of MSCs showed radial shockwave stimulation increased ALP activity but decreased formation of Oil Red-O-positive lipid droplets, indicating that shockwaves promote osteogenesis induction and block adipogenesis at the same time. No significant differences in the two cohorts of cells after chondrogenic induction. b Impact of shockwaves on MSC differentiation also reflected by mRNA levels of Runx-2 , Osterix , CEBP/α , PPARγ , Sox-9 , and Col-II . Quantitative PCR assay performed at least three times independently; representative result shown. *Statistically significant difference compared with control groups, P

    Techniques Used: ALP Assay, Activity Assay, Blocking Assay, Real-time Polymerase Chain Reaction

    35) Product Images from "Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells"

    Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

    Journal: Bone Research

    doi: 10.1038/s41413-018-0029-4

    ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P
    Figure Legend Snippet: ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P

    Techniques Used: Expressing, Western Blot, Transfection, Staining

    Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
    Figure Legend Snippet: Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

    Techniques Used: Mouse Assay, Injection, Expressing, Western Blot, Immunostaining, Staining, Labeling, Quantitative RT-PCR

    Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
    Figure Legend Snippet: Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

    Techniques Used: Staining, Immunostaining, Expressing, Western Blot, Concentration Assay, Radioactivity, Plasmid Preparation, Transduction, shRNA

    Alpl deficiency induces an elevation in extracellular ATP, which is internalized by MSCs and causes their dysfunction. a Extracellular ATP concentrations in Alpl +/+ and Alpl +/- MSC medium were assayed by a regular ATP concentration assay. b Apoptosis of Alpl +/+ and Alpl +/- MSCs was analyzed by flow cytometry. c , d Extracellular ATP concentrations were assayed 1 h after FBS deprivation and H 2 O 2 induction (50 mmol⋅L –1 ). e Extracellular ATP concentrations were assayed 48 h after transduction with different lentiviral vectors. f Expression levels of CD73 and CD39 in Alpl +/+ and Alpl +/- MSCs were analyzed by western blotting. g ATP concentrations were assayed after 2 min of treatment with 0, 1, 2, 5, 10 or 20 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in double-distilled water (dd H 2 O, pH 7.5). h ATP concentrations were assayed at 0 min, 2 min, 10 min and 30 min after treatment with 10 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in dd H 2 O. i , j Alpl +/+ and Alpl +/- MSCs were treated with 10 μmol⋅L –1 ATP in the presence or absence of 50 μmol⋅L –1 ethyl isopropyl amiloride (EIPA), 30 μmol⋅L –1 pyridoxal phosphate-6-azo (PPADS) or 100 μmol⋅L –1 suramin (Sur), and the intracellular ATP concentrations were assayed 1 h after treatment. k Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in the different lentivirus transduction groups. l Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentivirus transduction groups treated with 100 μmol⋅L –1 suramin and 50 μmol⋅L –1 EIPA. m, n Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP, and the ageing-specific genes were analyzed after 48 h. Expression levels of Runx2, OCN and PPARγ were examined by western blotting on day 7 after induction. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
    Figure Legend Snippet: Alpl deficiency induces an elevation in extracellular ATP, which is internalized by MSCs and causes their dysfunction. a Extracellular ATP concentrations in Alpl +/+ and Alpl +/- MSC medium were assayed by a regular ATP concentration assay. b Apoptosis of Alpl +/+ and Alpl +/- MSCs was analyzed by flow cytometry. c , d Extracellular ATP concentrations were assayed 1 h after FBS deprivation and H 2 O 2 induction (50 mmol⋅L –1 ). e Extracellular ATP concentrations were assayed 48 h after transduction with different lentiviral vectors. f Expression levels of CD73 and CD39 in Alpl +/+ and Alpl +/- MSCs were analyzed by western blotting. g ATP concentrations were assayed after 2 min of treatment with 0, 1, 2, 5, 10 or 20 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in double-distilled water (dd H 2 O, pH 7.5). h ATP concentrations were assayed at 0 min, 2 min, 10 min and 30 min after treatment with 10 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in dd H 2 O. i , j Alpl +/+ and Alpl +/- MSCs were treated with 10 μmol⋅L –1 ATP in the presence or absence of 50 μmol⋅L –1 ethyl isopropyl amiloride (EIPA), 30 μmol⋅L –1 pyridoxal phosphate-6-azo (PPADS) or 100 μmol⋅L –1 suramin (Sur), and the intracellular ATP concentrations were assayed 1 h after treatment. k Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in the different lentivirus transduction groups. l Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentivirus transduction groups treated with 100 μmol⋅L –1 suramin and 50 μmol⋅L –1 EIPA. m, n Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP, and the ageing-specific genes were analyzed after 48 h. Expression levels of Runx2, OCN and PPARγ were examined by western blotting on day 7 after induction. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

    Techniques Used: Concentration Assay, Flow Cytometry, Cytometry, Transduction, Expressing, Western Blot, Radioactivity

    Alpl controls the osteo-adipogenic balance in MSCs and prevents their senescence. a ALP activities and expression levels in Alpl +/+ and Alpl +/- MSCs were examined by an ALP activity assay and a western blotting analysis. b SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of first-passage MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months were examined by a western blotting analysis. d, e Expression levels of Runx2, OCN and PPAR-γ in MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months were examined by a western blotting analysis on day 7 after the osteogenic/adipogenic induction. f Downregulated Alpl expression in Alpl +/+ MSCs and upregulated expression in Alpl +/- MSCs by lentiviral vectors. Expression levels of the ageing-specific genes p16 and p53 in MSCs were examined by a western blotting analysis. g Expression levels of Runx2 and OCN were examined by a western blotting analysis on day 7 after the osteogenic induction. h HE/Masson’s trichrome staining and quantitative analysis revealed the formation of bone (B), bone marrow (BM) and collagen fiber (CF) around the HA/TCP (HA) carrier after the MSCs were implanted into nude mice. Scale bars, 200 μm. i PPAR-γ expression was examined on day 7 after the adipogenic induction by western blotting. j Alpl +/+ MSCs were treated with 50 μM H 2 O 2 for 24 h with or without the overexpression of Alpl ; then, the medium was replaced with normal medium, followed by incubation for another 24 h. Expression levels of p16 and p53 were examined by a western blotting analysis. k, l Alpl +/+ MSCs were treated with 50 μM H 2 O 2 for 24 h with or without Alpl overexpression, and then, the medium was changed to induction medium, followed by incubation for 7 d. Expression levels of Runx2, OCN and PPARγ were examined by a western blotting analysis. n = 6 per groups. The data are shown as the means ± s.d. of each independent experiment performed in triplicate. * P
    Figure Legend Snippet: Alpl controls the osteo-adipogenic balance in MSCs and prevents their senescence. a ALP activities and expression levels in Alpl +/+ and Alpl +/- MSCs were examined by an ALP activity assay and a western blotting analysis. b SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of first-passage MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months were examined by a western blotting analysis. d, e Expression levels of Runx2, OCN and PPAR-γ in MSCs from Alpl +/+ and Alpl +/- mice at 4 and 12 months were examined by a western blotting analysis on day 7 after the osteogenic/adipogenic induction. f Downregulated Alpl expression in Alpl +/+ MSCs and upregulated expression in Alpl +/- MSCs by lentiviral vectors. Expression levels of the ageing-specific genes p16 and p53 in MSCs were examined by a western blotting analysis. g Expression levels of Runx2 and OCN were examined by a western blotting analysis on day 7 after the osteogenic induction. h HE/Masson’s trichrome staining and quantitative analysis revealed the formation of bone (B), bone marrow (BM) and collagen fiber (CF) around the HA/TCP (HA) carrier after the MSCs were implanted into nude mice. Scale bars, 200 μm. i PPAR-γ expression was examined on day 7 after the adipogenic induction by western blotting. j Alpl +/+ MSCs were treated with 50 μM H 2 O 2 for 24 h with or without the overexpression of Alpl ; then, the medium was replaced with normal medium, followed by incubation for another 24 h. Expression levels of p16 and p53 were examined by a western blotting analysis. k, l Alpl +/+ MSCs were treated with 50 μM H 2 O 2 for 24 h with or without Alpl overexpression, and then, the medium was changed to induction medium, followed by incubation for 7 d. Expression levels of Runx2, OCN and PPARγ were examined by a western blotting analysis. n = 6 per groups. The data are shown as the means ± s.d. of each independent experiment performed in triplicate. * P

    Techniques Used: ALP Assay, Expressing, Activity Assay, Western Blot, Staining, Immunostaining, Mouse Assay, Over Expression, Incubation

    36) Product Images from "Distinct Roles of Glycogen Synthase Kinase (GSK)-3? and GSK-3? in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells *"

    Article Title: Distinct Roles of Glycogen Synthase Kinase (GSK)-3? and GSK-3? in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.019109

    The role of GSK-3α and GSK-3β in mediating cardiomyocyte differentiation of MSCs. A–D , MSCs were treated with Ad-shRNA-scramble ( Ad-sh-scramble ), Ad-shRNA-GSK-3α ( Ad-sh-GSK-3 α), or Ad-shRNA-GSK-3β ( Ad-sh-GSK-3 β) in the presence or absence of 5-Aza. A , protein expression of GSK-3α, GSK-3β, β-catenin, and GAPDH (internal control) was examined by immunoblot assays. A short exposure of an immunoblot is shown for β-catenin because the band with Ad-sh-GSK-3β was saturated after longer exposures. B , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, and β-actin (internal control) was examined by RT-PCR. C , protein expression of sarcomeric α-actinin, cTnI, GATA4, and β-actin (internal control) was examined by immunoblot assays. D , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, and GAPDH (internal control) was evaluated by RT-PCR. E and F , MSCs were transduced with Ad-GSK-3β together with Ad-sh-scramble or Ad-sh-GSK-3α. E , protein expression of GSK-3α, GSK-3β, β-catenin, and GAPDH (internal control) was evaluated by immunoblot assays. F , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, atrial natriuretic factor, and GAPDH was evaluated by RT-PCR. Please note that a smaller cycle number was used in RT-PCR to show that Ad-sh-GSK-3α enhances the effect of Ad-GSK-3β. In A–F , the results are representative of 3–4 experiments.
    Figure Legend Snippet: The role of GSK-3α and GSK-3β in mediating cardiomyocyte differentiation of MSCs. A–D , MSCs were treated with Ad-shRNA-scramble ( Ad-sh-scramble ), Ad-shRNA-GSK-3α ( Ad-sh-GSK-3 α), or Ad-shRNA-GSK-3β ( Ad-sh-GSK-3 β) in the presence or absence of 5-Aza. A , protein expression of GSK-3α, GSK-3β, β-catenin, and GAPDH (internal control) was examined by immunoblot assays. A short exposure of an immunoblot is shown for β-catenin because the band with Ad-sh-GSK-3β was saturated after longer exposures. B , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, and β-actin (internal control) was examined by RT-PCR. C , protein expression of sarcomeric α-actinin, cTnI, GATA4, and β-actin (internal control) was examined by immunoblot assays. D , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, and GAPDH (internal control) was evaluated by RT-PCR. E and F , MSCs were transduced with Ad-GSK-3β together with Ad-sh-scramble or Ad-sh-GSK-3α. E , protein expression of GSK-3α, GSK-3β, β-catenin, and GAPDH (internal control) was evaluated by immunoblot assays. F , mRNA expression of Flk-1, Nkx2.5, α-MHC, cTnC, atrial natriuretic factor, and GAPDH was evaluated by RT-PCR. Please note that a smaller cycle number was used in RT-PCR to show that Ad-sh-GSK-3α enhances the effect of Ad-GSK-3β. In A–F , the results are representative of 3–4 experiments.

    Techniques Used: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction

    37) Product Images from "Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells"

    Article Title: Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.338

    Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. ( a ) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. ( b ) MSCs were stained with γ -H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. ( c and d ) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. ( e and f ) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence
    Figure Legend Snippet: Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. ( a ) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. ( b ) MSCs were stained with γ -H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. ( c and d ) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. ( e and f ) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence

    Techniques Used: Irradiation, Staining, Immunofluorescence, Fluorescence, Microscopy, FACS

    38) Product Images from "Dexamethasone-Activated MSCs Release MVs for Stimulating Osteogenic Response"

    Article Title: Dexamethasone-Activated MSCs Release MVs for Stimulating Osteogenic Response

    Journal: Stem Cells International

    doi: 10.1155/2018/7231739

    Characterization of MSC-MVs (n-MVs or DXM-MVs). MSCs were activated by treatment with 10 −8 , 10 −7 , and 10 −6 M DXM. (a) Size distribution and concentration of MSC-MVs detected by NTA analysis. (b) Typical morphology of MSC-MVs investigated by a transmission electron microscope. (c) Flow cytometric analysis showing the expression of MSC-specific marker CD90 in MSC-MVs.
    Figure Legend Snippet: Characterization of MSC-MVs (n-MVs or DXM-MVs). MSCs were activated by treatment with 10 −8 , 10 −7 , and 10 −6 M DXM. (a) Size distribution and concentration of MSC-MVs detected by NTA analysis. (b) Typical morphology of MSC-MVs investigated by a transmission electron microscope. (c) Flow cytometric analysis showing the expression of MSC-specific marker CD90 in MSC-MVs.

    Techniques Used: Concentration Assay, Transmission Assay, Microscopy, Flow Cytometry, Expressing, Marker

    39) Product Images from "Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells"

    Article Title: Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089882

    Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo. Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P > 0.01.
    Figure Legend Snippet: Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo. Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P > 0.01.

    Techniques Used: Derivative Assay, In Vivo, In Vitro, MTT Assay, Functional Assay, Incubation, Injection, Mouse Assay, Transplantation Assay, Inhibition

    40) Product Images from "Characterisation of Cultured Mesothelial Cells Derived from the Murine Adult Omentum"

    Article Title: Characterisation of Cultured Mesothelial Cells Derived from the Murine Adult Omentum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158997

    Analysis of osteogenic and adipogenic potential. Using Alizarin S red staining, Calcium deposits could be detected in P13 MCs (B) and P21 MSCs (D), indicating osteogenic differentiation, while cells under control conditions failed to exhibit the deposits (A, C). Fat droplet accumulation could be detected in P5 MCs (F) and, slightly more pronounced in MSCs (H). Control conditions showed no generation of fat droplets (E, G). Expression analysis by qPCR revealed that the osteogenic marker Sparc was up-regulated in the earlier passages (I), with a significant 6-fold change in P13 MCs. A significant 3.7-fold increase in expression was observed in P5 MCs for the adipogenic gene PPARγ (K). A Student’s t-test was used to calculate significance. Scale bars are 30 μM.
    Figure Legend Snippet: Analysis of osteogenic and adipogenic potential. Using Alizarin S red staining, Calcium deposits could be detected in P13 MCs (B) and P21 MSCs (D), indicating osteogenic differentiation, while cells under control conditions failed to exhibit the deposits (A, C). Fat droplet accumulation could be detected in P5 MCs (F) and, slightly more pronounced in MSCs (H). Control conditions showed no generation of fat droplets (E, G). Expression analysis by qPCR revealed that the osteogenic marker Sparc was up-regulated in the earlier passages (I), with a significant 6-fold change in P13 MCs. A significant 3.7-fold increase in expression was observed in P5 MCs for the adipogenic gene PPARγ (K). A Student’s t-test was used to calculate significance. Scale bars are 30 μM.

    Techniques Used: Staining, Expressing, Real-time Polymerase Chain Reaction, Marker

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  • 96
    Millipore hmscs
    (A) <t>Micropatterned</t> <t>hMSCs</t> stained against F-actin after 24 hours incubation. Triangular and square shaped cells result in formation of large stress fibres on the cell perimeter spanning from on edge to another, while round cells show a cortical F-actin network with smaller fibres. (B) Micropatterned cells stained for myosin IIa show a similar trend in myosin fibre formation as observed by the cell shape dependent changes of actin cytoskeleton. The separate images as well as overlay of pan-myosin IIa (green) as well as phospho-myosin IIa (red) is shown. (C) Immunofluorescence intensity heat maps of > 30 micropatterned single hMSCs stained for phosphorylated-myosin IIa and pan-myosin IIa. Higher intensity is represented by brighter colours. Scale bar = 20 µm.
    Hmscs, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore spio mscs
    CMR imaging. Notes: ( A ) Representative in vivo CMR imaging of injected 5×10 5 unlabeled <t>MSCs,</t> ( B ) living <t>SPIO-MSCs,</t> ( C ) dead SPIO-MSCs, and ( D ) SPIO (0.6 μL Resovist) in swine heart. Red arrows in figures B – D indicate the signal void corresponding to the injection sites. ( E ) Quantitative analysis of signal intensity. ⊿SI=[(SI−SIunlabeled MSCs)/SIunlabeled MSCs]×100%. Abbreviations: CMR, cardiac magnetic resonance; LV, left ventricle; MSCs, mesenchymal stem cells; NS, not significant; RV, right ventricle; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; SI, signal intensity.
    Spio Mscs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Micropatterned hMSCs stained against F-actin after 24 hours incubation. Triangular and square shaped cells result in formation of large stress fibres on the cell perimeter spanning from on edge to another, while round cells show a cortical F-actin network with smaller fibres. (B) Micropatterned cells stained for myosin IIa show a similar trend in myosin fibre formation as observed by the cell shape dependent changes of actin cytoskeleton. The separate images as well as overlay of pan-myosin IIa (green) as well as phospho-myosin IIa (red) is shown. (C) Immunofluorescence intensity heat maps of > 30 micropatterned single hMSCs stained for phosphorylated-myosin IIa and pan-myosin IIa. Higher intensity is represented by brighter colours. Scale bar = 20 µm.

    Journal: The Analyst

    Article Title: High resolution Raman spectroscopy mapping of stem cell micropatterns †

    doi: 10.1039/c4an02346c

    Figure Lengend Snippet: (A) Micropatterned hMSCs stained against F-actin after 24 hours incubation. Triangular and square shaped cells result in formation of large stress fibres on the cell perimeter spanning from on edge to another, while round cells show a cortical F-actin network with smaller fibres. (B) Micropatterned cells stained for myosin IIa show a similar trend in myosin fibre formation as observed by the cell shape dependent changes of actin cytoskeleton. The separate images as well as overlay of pan-myosin IIa (green) as well as phospho-myosin IIa (red) is shown. (C) Immunofluorescence intensity heat maps of > 30 micropatterned single hMSCs stained for phosphorylated-myosin IIa and pan-myosin IIa. Higher intensity is represented by brighter colours. Scale bar = 20 µm.

    Article Snippet: A total of 8–12 micropatterned single hMSCs from independent cultures for each micro-island shape were measured. hMSCs were fixed with 4% (v/v) formalin in dH2 O (Sigma) for 15 minutes at room temperature, washed PBS three times, and stored at 4 °C for maximum 3 days before being analysed.

    Techniques: Staining, Incubation, Immunofluorescence

    Micropatterned hMSCs after 24 hours incubation. Cells adapt to the underlining shape of the FN micro-islands resulting into triangular, square, and circular shaped cells. The islands have an identical cell adhesion area of 1350 µm 2 but a different cellular architecture.

    Journal: The Analyst

    Article Title: High resolution Raman spectroscopy mapping of stem cell micropatterns †

    doi: 10.1039/c4an02346c

    Figure Lengend Snippet: Micropatterned hMSCs after 24 hours incubation. Cells adapt to the underlining shape of the FN micro-islands resulting into triangular, square, and circular shaped cells. The islands have an identical cell adhesion area of 1350 µm 2 but a different cellular architecture.

    Article Snippet: A total of 8–12 micropatterned single hMSCs from independent cultures for each micro-island shape were measured. hMSCs were fixed with 4% (v/v) formalin in dH2 O (Sigma) for 15 minutes at room temperature, washed PBS three times, and stored at 4 °C for maximum 3 days before being analysed.

    Techniques: Incubation

    (A) Representative immunofluoresence images of micropatterned hMSCs stained against collagen I. (B) Immunofluoresence intensity heatmaps of triangular, square, and circular shaped micropatterned hMSCs stained against collagen I illustrate the previously observed localisation dependent signal intensity and overall collagen I abundance across the whole cell population quantitatively. Scale bar = 20 µm. (C) Immunofluorescence image quantification of the average signal intensity of micropatterned hMSCs stained against collagen I.

    Journal: The Analyst

    Article Title: High resolution Raman spectroscopy mapping of stem cell micropatterns †

    doi: 10.1039/c4an02346c

    Figure Lengend Snippet: (A) Representative immunofluoresence images of micropatterned hMSCs stained against collagen I. (B) Immunofluoresence intensity heatmaps of triangular, square, and circular shaped micropatterned hMSCs stained against collagen I illustrate the previously observed localisation dependent signal intensity and overall collagen I abundance across the whole cell population quantitatively. Scale bar = 20 µm. (C) Immunofluorescence image quantification of the average signal intensity of micropatterned hMSCs stained against collagen I.

    Article Snippet: A total of 8–12 micropatterned single hMSCs from independent cultures for each micro-island shape were measured. hMSCs were fixed with 4% (v/v) formalin in dH2 O (Sigma) for 15 minutes at room temperature, washed PBS three times, and stored at 4 °C for maximum 3 days before being analysed.

    Techniques: Staining, Immunofluorescence

    Fabrication and assembly of the OMA hydrogel beads and hMSC-laden OMA microgels. (a) Schematic depicting i) OMA bead fabrication and ii) Ca-crosslinked OMA bead. (b) Fabrication of assembled letters of manually arranged OMA beads connected by photocrosslinking. i) Ca-crosslinked OMA beads were manually arranged on a glass plate and then assembled under UV light. ii) Physically linked OMA beads in the letter C were mechanically stable. iii) Methacrylate groups were photocrosslinked under UV light between the OMA bead units to stabilize the resulting assembly. iv) Beads were manually arranged to form the letter E on a glass plate. v) OMA beads joined together via photocrosslinking could be lifted up from the glass plate. vi) Individual OMA beads detached from non-UV irradiated OMA bead samples. The scale bars indicate 10 mm. (c) i) Schematic diagram of coaxial airflow-induced microgel generator and ii) representative photograph of hMSC-laden OMA microgels. (d) Live/Dead staining of encapsulated hMSCs in OMA microgels at day 0. Green color indicates vital cells and red color indicates dead cells. (e) Live/Dead images of hMSC-laden microgels after 4 weeks culture before (i) and after (ii) assembly under UV light. The scale bars indicate 200 μm.

    Journal: Materials today. Chemistry

    Article Title: Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues

    doi: 10.1016/j.mtchem.2018.11.009

    Figure Lengend Snippet: Fabrication and assembly of the OMA hydrogel beads and hMSC-laden OMA microgels. (a) Schematic depicting i) OMA bead fabrication and ii) Ca-crosslinked OMA bead. (b) Fabrication of assembled letters of manually arranged OMA beads connected by photocrosslinking. i) Ca-crosslinked OMA beads were manually arranged on a glass plate and then assembled under UV light. ii) Physically linked OMA beads in the letter C were mechanically stable. iii) Methacrylate groups were photocrosslinked under UV light between the OMA bead units to stabilize the resulting assembly. iv) Beads were manually arranged to form the letter E on a glass plate. v) OMA beads joined together via photocrosslinking could be lifted up from the glass plate. vi) Individual OMA beads detached from non-UV irradiated OMA bead samples. The scale bars indicate 10 mm. (c) i) Schematic diagram of coaxial airflow-induced microgel generator and ii) representative photograph of hMSC-laden OMA microgels. (d) Live/Dead staining of encapsulated hMSCs in OMA microgels at day 0. Green color indicates vital cells and red color indicates dead cells. (e) Live/Dead images of hMSC-laden microgels after 4 weeks culture before (i) and after (ii) assembly under UV light. The scale bars indicate 200 μm.

    Article Snippet: [ ] To fabricate hMSC-laden OMA microgels, hMSCs were expanded in growth media consisting of DMEM-LG with 10 % FBS (Sigma), 1 % P/S and 10 ng/ml FGF-2 (R & D) and suspended in OMA solution (passage 3, 2×106 cells/ml). hMSC-suspended OMA solutions were loaded into an 3-ml syringe, and then the syringe was connected to a coaxial microdroplet generator, designed in our laboratory ( and ).

    Techniques: Irradiation, Staining

    CMR imaging. Notes: ( A ) Representative in vivo CMR imaging of injected 5×10 5 unlabeled MSCs, ( B ) living SPIO-MSCs, ( C ) dead SPIO-MSCs, and ( D ) SPIO (0.6 μL Resovist) in swine heart. Red arrows in figures B – D indicate the signal void corresponding to the injection sites. ( E ) Quantitative analysis of signal intensity. ⊿SI=[(SI−SIunlabeled MSCs)/SIunlabeled MSCs]×100%. Abbreviations: CMR, cardiac magnetic resonance; LV, left ventricle; MSCs, mesenchymal stem cells; NS, not significant; RV, right ventricle; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; SI, signal intensity.

    Journal: International Journal of Nanomedicine

    Article Title: Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium

    doi: 10.2147/IJN.S77858

    Figure Lengend Snippet: CMR imaging. Notes: ( A ) Representative in vivo CMR imaging of injected 5×10 5 unlabeled MSCs, ( B ) living SPIO-MSCs, ( C ) dead SPIO-MSCs, and ( D ) SPIO (0.6 μL Resovist) in swine heart. Red arrows in figures B – D indicate the signal void corresponding to the injection sites. ( E ) Quantitative analysis of signal intensity. ⊿SI=[(SI−SIunlabeled MSCs)/SIunlabeled MSCs]×100%. Abbreviations: CMR, cardiac magnetic resonance; LV, left ventricle; MSCs, mesenchymal stem cells; NS, not significant; RV, right ventricle; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; SI, signal intensity.

    Article Snippet: To determine the differentiation of myocardium-like cells, MSCs and SPIO-MSCs were cultured in culture medium with added 5-aza-2′-deoxycytidine (5-aza-C; Sigma-Aldrich Co) for 24 hours.

    Techniques: Imaging, In Vivo, Injection, Incubation

    Release rate of intracellular iron in vitro. Notes: ( A ) Prussian blue staining of SPIO-MSCs at different times after SPIO labeling (all figures in A are at 50 μm in size range). ( B ) Quantitative analysis showing that the intracellular mean iron load decreased continuously over time after magnetic labeling. ( C ) Electron microscopic images of MSCs at 4 days after labeling showed the release of iron dense particles (red arrow) from the cytoplasm. Abbreviations: d, days; SPIO, superparamagnetic iron oxide; MSCs, mesenchymal stem cells; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide.

    Journal: International Journal of Nanomedicine

    Article Title: Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium

    doi: 10.2147/IJN.S77858

    Figure Lengend Snippet: Release rate of intracellular iron in vitro. Notes: ( A ) Prussian blue staining of SPIO-MSCs at different times after SPIO labeling (all figures in A are at 50 μm in size range). ( B ) Quantitative analysis showing that the intracellular mean iron load decreased continuously over time after magnetic labeling. ( C ) Electron microscopic images of MSCs at 4 days after labeling showed the release of iron dense particles (red arrow) from the cytoplasm. Abbreviations: d, days; SPIO, superparamagnetic iron oxide; MSCs, mesenchymal stem cells; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide.

    Article Snippet: To determine the differentiation of myocardium-like cells, MSCs and SPIO-MSCs were cultured in culture medium with added 5-aza-2′-deoxycytidine (5-aza-C; Sigma-Aldrich Co) for 24 hours.

    Techniques: In Vitro, Staining, Labeling, Incubation

    Proliferation, viability, and differentiation capacity of SPIO-MSCs. Notes: ( A ) Proliferation rate of SPIO-MSCs. ( B ) Viability rate of SPIO-MSCs. ( C ) Oil red O staining for adipogenic differentiation (both images in C are at 100 μm in size range). ( D ) RT-PCR for myocardial gene cTnT, Desmin , and α-cardiac actin . ( E ) Quantitative gene expression analysis. SPIO labeling did not affect the proliferation, viability, and differentiation capacity of MSCs. Abbreviations: MSCs, mesenchymal stem cells; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; RT-PCR, real-time quantitative polymerase chain reaction; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; NS, not significant; NC, negative control; PC, positive control.

    Journal: International Journal of Nanomedicine

    Article Title: Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium

    doi: 10.2147/IJN.S77858

    Figure Lengend Snippet: Proliferation, viability, and differentiation capacity of SPIO-MSCs. Notes: ( A ) Proliferation rate of SPIO-MSCs. ( B ) Viability rate of SPIO-MSCs. ( C ) Oil red O staining for adipogenic differentiation (both images in C are at 100 μm in size range). ( D ) RT-PCR for myocardial gene cTnT, Desmin , and α-cardiac actin . ( E ) Quantitative gene expression analysis. SPIO labeling did not affect the proliferation, viability, and differentiation capacity of MSCs. Abbreviations: MSCs, mesenchymal stem cells; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; RT-PCR, real-time quantitative polymerase chain reaction; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; NS, not significant; NC, negative control; PC, positive control.

    Article Snippet: To determine the differentiation of myocardium-like cells, MSCs and SPIO-MSCs were cultured in culture medium with added 5-aza-2′-deoxycytidine (5-aza-C; Sigma-Aldrich Co) for 24 hours.

    Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Labeling, Incubation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    In vivo magnetic resonance imaging. Notes: ( A ) In vivo magnetic resonance imaging of tube-containing gel with 5×10 5 unlabeled MSCs, living SPIO-MSCs, dead SPIO-MSCs, and SPIO alone (0.6 μL Resovist) without MSCs, respectively. ( B ) Quantitative analysis of signal intensity. Equation for SI is ⊿SI = [(SI−SI unlabeled MSCs)/SI unlabeled MSCs] ×100%. Abbreviations: MSCs, mesenchymal stem cells; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; SI, signal intensity; FSE, fast spin echo; T 1 WI, T1 weighted imaging; T 2 WI, T2 weighted imaging; T 2 *WI, T2 star weighted imaging.

    Journal: International Journal of Nanomedicine

    Article Title: Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium

    doi: 10.2147/IJN.S77858

    Figure Lengend Snippet: In vivo magnetic resonance imaging. Notes: ( A ) In vivo magnetic resonance imaging of tube-containing gel with 5×10 5 unlabeled MSCs, living SPIO-MSCs, dead SPIO-MSCs, and SPIO alone (0.6 μL Resovist) without MSCs, respectively. ( B ) Quantitative analysis of signal intensity. Equation for SI is ⊿SI = [(SI−SI unlabeled MSCs)/SI unlabeled MSCs] ×100%. Abbreviations: MSCs, mesenchymal stem cells; SPIO, superparamagnetic iron oxide; SPIO-MSCs, mesenchymal stem cells incubated with superparamagnetic iron oxide; SI, signal intensity; FSE, fast spin echo; T 1 WI, T1 weighted imaging; T 2 WI, T2 weighted imaging; T 2 *WI, T2 star weighted imaging.

    Article Snippet: To determine the differentiation of myocardium-like cells, MSCs and SPIO-MSCs were cultured in culture medium with added 5-aza-2′-deoxycytidine (5-aza-C; Sigma-Aldrich Co) for 24 hours.

    Techniques: In Vivo, Magnetic Resonance Imaging, Incubation, Imaging

    Morphological change of MSCs with or without the exposure to CART . Particular changes in morphology happened to MSCs with the exposure to CART. Similar to the situation of the bFGF/EGF-treated group, several MSCs incubated with CART for 3 days became shorter and nucleus-convergent. They subsequently evolved to display round cell bodies with long axons in 6 days. In the control group, mesenchmal stem cells nearly kept stable in the appearance within the 6 days of, observation (scale bar = 50 uM).

    Journal: BMC Neuroscience

    Article Title: Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    doi: 10.1186/1471-2202-12-67

    Figure Lengend Snippet: Morphological change of MSCs with or without the exposure to CART . Particular changes in morphology happened to MSCs with the exposure to CART. Similar to the situation of the bFGF/EGF-treated group, several MSCs incubated with CART for 3 days became shorter and nucleus-convergent. They subsequently evolved to display round cell bodies with long axons in 6 days. In the control group, mesenchmal stem cells nearly kept stable in the appearance within the 6 days of, observation (scale bar = 50 uM).

    Article Snippet: As a positive control, MSCs were differentiated by DMEM/10 ug.ml-1 EGF/10 ug.ml-1 bFGF(Sigma, USA).

    Techniques: Incubation

    Differential spectrum of MSCs determined by Immunofluorescence . Nestin, GFAP, MAP-2 and NeuN were involved to detect neural progenitors, glial cells, and mature neurons. In the CART treated group (A), the percentage of Nestin (green) positive cells occupied 25.4 ± 2.1% in Day 3 and 47.1 ± 1.9% in Day 6. (B) The NeuN (green) positive rate was 32.1 ± 2.3% in 3 days and 40.3 ± 2.7% in 6 days. The differentiation ratio resembled that of bFGF/EGF. (C) MAP-2 (red) was detected in 30.8 ± 4.7% and 41.2 ± 3.1% of all cells in Day 3 and Day 6. (D) GFAP (green) ranks 20.5 ± 2.5% in 3 days and 21.3 ± 2.2% in 6 days. The converted rate resembled that of bFGF/EGF. The untreated group was used as a control..

    Journal: BMC Neuroscience

    Article Title: Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    doi: 10.1186/1471-2202-12-67

    Figure Lengend Snippet: Differential spectrum of MSCs determined by Immunofluorescence . Nestin, GFAP, MAP-2 and NeuN were involved to detect neural progenitors, glial cells, and mature neurons. In the CART treated group (A), the percentage of Nestin (green) positive cells occupied 25.4 ± 2.1% in Day 3 and 47.1 ± 1.9% in Day 6. (B) The NeuN (green) positive rate was 32.1 ± 2.3% in 3 days and 40.3 ± 2.7% in 6 days. The differentiation ratio resembled that of bFGF/EGF. (C) MAP-2 (red) was detected in 30.8 ± 4.7% and 41.2 ± 3.1% of all cells in Day 3 and Day 6. (D) GFAP (green) ranks 20.5 ± 2.5% in 3 days and 21.3 ± 2.2% in 6 days. The converted rate resembled that of bFGF/EGF. The untreated group was used as a control..

    Article Snippet: As a positive control, MSCs were differentiated by DMEM/10 ug.ml-1 EGF/10 ug.ml-1 bFGF(Sigma, USA).

    Techniques: Immunofluorescence

    Founctional test of the differentiated MSCs . Cholinergic neurons and dopaminergic neurons were investigated by double labeled immunofluorescence with the antibodies against ChAT (green) and NeuN as well as TH (green) and NeuN (red) with the purpose of finding founctional neurons. NeuN positive cells co-expressed with ChAT (A) or TH (B) in the CART-treated group and the bFGF/EGF-incubated group. (C) The transferred neurons exhibited cytoplasmic dark blue praticles and light blue nucleus by Nissl stain. MSCs devoid of CART displayed dark blue nucleus and light blue cytoplasm (scale bar = 50 uM).

    Journal: BMC Neuroscience

    Article Title: Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    doi: 10.1186/1471-2202-12-67

    Figure Lengend Snippet: Founctional test of the differentiated MSCs . Cholinergic neurons and dopaminergic neurons were investigated by double labeled immunofluorescence with the antibodies against ChAT (green) and NeuN as well as TH (green) and NeuN (red) with the purpose of finding founctional neurons. NeuN positive cells co-expressed with ChAT (A) or TH (B) in the CART-treated group and the bFGF/EGF-incubated group. (C) The transferred neurons exhibited cytoplasmic dark blue praticles and light blue nucleus by Nissl stain. MSCs devoid of CART displayed dark blue nucleus and light blue cytoplasm (scale bar = 50 uM).

    Article Snippet: As a positive control, MSCs were differentiated by DMEM/10 ug.ml-1 EGF/10 ug.ml-1 bFGF(Sigma, USA).

    Techniques: Labeling, Immunofluorescence, Incubation, Staining