hmscs  (Millipore)

 
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    Name:
    Human Mesenchymal Stem Cells
    Description:

    Catalog Number:
    C-12974
    Price:
    None
    Applications:
    Mesenchymal Stem Cells (MSC), also termed Mesenchymal Stromal Cells, are multipotent cells that can differentiate into a variety of cell types and have the capacity for self renewal. MSC have been shown to differentiate in vitro or in vivo into adipocytes, chondrocytes, osteoblasts, myocytes, neurons, hepatocytes, and pancreatic islet cells. Optimized PromoCell media are available to support both the growth of MSC and their differentiation into several different lineages. Recent experiments suggest that differentiation capabilities into diverse cell types vary between MSC of different origin.Human Mesenchymal Stem Cells (hMSC-BM) are harvested from normal human bone marrow from individual donors and are provided in a cryopreserved format. The cells are tested for their ability to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts. The cells show a verified marker expression profile that complies with ISCT recommendations, providing well characterized cells (Cytotherapy (2006) Vol. 8, No. 4, 315-317).
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    Millipore hmscs
    Human Mesenchymal Stem Cells

    https://www.bioz.com/result/hmscs/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hmscs - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues"

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

    Journal: Materials today. Chemistry

    doi: 10.1016/j.mtchem.2018.11.009

    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.
    Figure Legend 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.

    Techniques Used: Irradiation, Staining

    2) Product Images from "Combination of Human Mesenchymal Stem Cells and Repetitive Transcranial Magnetic Stimulation Enhances Neurological Recovery of 6-Hydroxydopamine Model of Parkinsonian’s Disease"

    Article Title: Combination of Human Mesenchymal Stem Cells and Repetitive Transcranial Magnetic Stimulation Enhances Neurological Recovery of 6-Hydroxydopamine Model of Parkinsonian’s Disease

    Journal: Tissue Engineering and Regenerative Medicine

    doi: 10.1007/s13770-019-00233-8

    Recruitment of hMSCs to the ipsilateral hemisphere. A PKH26-labeled hMSCs, which were transplanted into the cisterna magna, are recruited to the ST at 4 wpt. Right the figures B is a higher magnification of the ST area demarcated with a rectangle in ( A ) is shown. C Recruitment of hMSCs to the SN area. PKH26-labeled hMSCs are recruited to the ipsilateral SN of PD rats at 4 wpt. D Is higher magnification of the SN area demarcated with a rectangle in ( C ). E – G PKH26 and DAPI double labeling in the ipsilateral SN. PKH26-labeled hMSCs are recruited to the SN at 4 week after hMSC transplantation. C – E DAPI labeled cells in the SN ( F ). A few PKH26-labeled hMSCs co-localized with DAPI (arrows) in the ipsilateral SN ( G ) of PD rats at 4 week after transplantation. ST striatum, SN substantia nigra
    Figure Legend Snippet: Recruitment of hMSCs to the ipsilateral hemisphere. A PKH26-labeled hMSCs, which were transplanted into the cisterna magna, are recruited to the ST at 4 wpt. Right the figures B is a higher magnification of the ST area demarcated with a rectangle in ( A ) is shown. C Recruitment of hMSCs to the SN area. PKH26-labeled hMSCs are recruited to the ipsilateral SN of PD rats at 4 wpt. D Is higher magnification of the SN area demarcated with a rectangle in ( C ). E – G PKH26 and DAPI double labeling in the ipsilateral SN. PKH26-labeled hMSCs are recruited to the SN at 4 week after hMSC transplantation. C – E DAPI labeled cells in the SN ( F ). A few PKH26-labeled hMSCs co-localized with DAPI (arrows) in the ipsilateral SN ( G ) of PD rats at 4 week after transplantation. ST striatum, SN substantia nigra

    Techniques Used: Labeling, Transplantation Assay

    3) Product Images from "High resolution Raman spectroscopy mapping of stem cell micropatterns †"

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

    Journal: The Analyst

    doi: 10.1039/c4an02346c

    (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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Staining, Immunofluorescence

    4) Product Images from "cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo"

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0711190105

    db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P
    Figure Legend Snippet: db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P

    Techniques Used: In Vivo, Cell Culture, Mouse Assay, Derivative Assay

    PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P
    Figure Legend Snippet: PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P

    Techniques Used: Activation Assay, In Vitro, ALP Assay, Expressing

    Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.
    Figure Legend Snippet: Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.

    Techniques Used: Expressing

    db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P
    Figure Legend Snippet: db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering"

    Article Title: Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055451

    Histological cross-sections of scaffolds seeded with BCH and hMSCs stained by H E and Alcian blue at different days of culture in differentiation medium.
    Figure Legend Snippet: Histological cross-sections of scaffolds seeded with BCH and hMSCs stained by H E and Alcian blue at different days of culture in differentiation medium.

    Techniques Used: Staining

    DNA assay on the scaffolds seeded with BCH and hMSCs in differentiation medium. Significant differences between each cell type at different time points were found for p
    Figure Legend Snippet: DNA assay on the scaffolds seeded with BCH and hMSCs in differentiation medium. Significant differences between each cell type at different time points were found for p

    Techniques Used:

    6) Product Images from "A Newly Identified Mechanism Involved in Regulation of Human Mesenchymal Stem Cells by Fibrous Substrate Stiffness"

    Article Title: A Newly Identified Mechanism Involved in Regulation of Human Mesenchymal Stem Cells by Fibrous Substrate Stiffness

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2016.06.034

    Detection of proteins and mRNA transcripts in hMSCs cultured on fibrous substrates. (A) Western blots of proteins extracted from cells on 75PLLA treated with or without AKT Inhibitor IV. (B) Protein and transcript expression of MIF from cells on 75PLLA
    Figure Legend Snippet: Detection of proteins and mRNA transcripts in hMSCs cultured on fibrous substrates. (A) Western blots of proteins extracted from cells on 75PLLA treated with or without AKT Inhibitor IV. (B) Protein and transcript expression of MIF from cells on 75PLLA

    Techniques Used: Cell Culture, Western Blot, Expressing

    Cell morphology and proliferation regulated by stiffness of fibrous substrates. (A) Cytoskeleton of hMSCs on PLLA, 65PLLA, and 75PLLA after 3 days of culture stained by phalloidin. Scale bar = 100 μm. (B) Aspect ratios of hMSCs cultured on different
    Figure Legend Snippet: Cell morphology and proliferation regulated by stiffness of fibrous substrates. (A) Cytoskeleton of hMSCs on PLLA, 65PLLA, and 75PLLA after 3 days of culture stained by phalloidin. Scale bar = 100 μm. (B) Aspect ratios of hMSCs cultured on different

    Techniques Used: Staining, Cell Culture

    Identification of regulatory molecules involved in a substrate stiffness-induced mechanism. (A) Detection of signaling molecules and transcription factors in hMSCs cultured on PLLA and 75PLLA by western blotting of whole cell lysate. (B) Confocal laser
    Figure Legend Snippet: Identification of regulatory molecules involved in a substrate stiffness-induced mechanism. (A) Detection of signaling molecules and transcription factors in hMSCs cultured on PLLA and 75PLLA by western blotting of whole cell lysate. (B) Confocal laser

    Techniques Used: Cell Culture, Western Blot

    Ostegenic differentiation of hMSCs cultured on PLLA and 75PLLA. Cells were cultured in basal medium for 7 days before induced in osteogenic differentiation medium for additional 14 days. (A-C) Expression levels of mRNA transcripts of bone-associated markers
    Figure Legend Snippet: Ostegenic differentiation of hMSCs cultured on PLLA and 75PLLA. Cells were cultured in basal medium for 7 days before induced in osteogenic differentiation medium for additional 14 days. (A-C) Expression levels of mRNA transcripts of bone-associated markers

    Techniques Used: Cell Culture, Expressing

    7) Product Images from "Transplantation of Heterospheroids of Islet Cells and Mesenchymal Stem Cells for Effective Angiogenesis and Antiapoptosis"

    Article Title: Transplantation of Heterospheroids of Islet Cells and Mesenchymal Stem Cells for Effective Angiogenesis and Antiapoptosis

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2014.0022

    ICs and hMSC localization in the portal vein of the liver. Before transplantation, ICs and hMSCs were labeled with PKH26 ( red ) and PKH67 ( green ), respectively. The livers were harvested 7 days after transplantation. Arrows indicate the ICs localized with hMSCs in the liver. Blue indicates nuclei stained with DAPI. Scale bars=50 μm. The percentages of both PKH67-positive (hMSC) and PKH26-positive (IC) clusters were evaluated from total clusters found in each liver ( lower panel ). * p
    Figure Legend Snippet: ICs and hMSC localization in the portal vein of the liver. Before transplantation, ICs and hMSCs were labeled with PKH26 ( red ) and PKH67 ( green ), respectively. The livers were harvested 7 days after transplantation. Arrows indicate the ICs localized with hMSCs in the liver. Blue indicates nuclei stained with DAPI. Scale bars=50 μm. The percentages of both PKH67-positive (hMSC) and PKH26-positive (IC) clusters were evaluated from total clusters found in each liver ( lower panel ). * p

    Techniques Used: Transplantation Assay, Labeling, Staining

    8) Product Images from "Lineage-Specific Exosomes Could Override Extracellular Matrix Mediated Human Mesenchymal Stem Cell Differentiation"

    Article Title: Lineage-Specific Exosomes Could Override Extracellular Matrix Mediated Human Mesenchymal Stem Cell Differentiation

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2018.08.027

    Characterization of extracellular matrix and exosome internalization. Normal human osteoblasts (NHO) and pre-adipocytes were differentiated to either osteoblasts or adipocytes on glass coverslips, as described in methods. The lysed cells were aspirated and the stiffness of the selected area (50 µm x 50 µm) deposited extracellular matrix (ECM) was examined under atomic force microscope (AFM). Young’s modulus (A) presented as mean ± S.D. for osteoblast and adipocyte ECM were calculated from 10 randomly selected regions using Hertz model. The extracted ECM was quantitated for specific proteins by ELISA. Type I collagen, fibronectin, laminin and Type IV collagen were measured for three independent extractions and presented as mean ± S.D. (B). Adhered human mesenchymal stem cells (hMSCs) were incubated with either labeled exosomes or free-dye for 24 h, as described in Materials and Methods section. Following incubation, the cells were washed with PBS, fixed with paraformaldehyde and fluorescence intensity was measured in a plate reader. Exosome uptake is presented as mean ± S.D. of fluorescence intensity of osteoblast (Os-Exo), adipocyte (Ad-Exo) and hMSC (hMSC-Exo). The hMSCs were supplemented with labeled exosomes for 2 h either at 37°C (solid bar) in the presence of 5µM (vertical lines bars) and 10µM (horizontal lines bars) of chlorpromazine. While incubation of hMSCs with exosomes at 4°C (slanted line bars) inhibited the uptake of exosomes. One-way ANOVA was used to evaluate statistical significance between groups (*p ≤ 0.005).
    Figure Legend Snippet: Characterization of extracellular matrix and exosome internalization. Normal human osteoblasts (NHO) and pre-adipocytes were differentiated to either osteoblasts or adipocytes on glass coverslips, as described in methods. The lysed cells were aspirated and the stiffness of the selected area (50 µm x 50 µm) deposited extracellular matrix (ECM) was examined under atomic force microscope (AFM). Young’s modulus (A) presented as mean ± S.D. for osteoblast and adipocyte ECM were calculated from 10 randomly selected regions using Hertz model. The extracted ECM was quantitated for specific proteins by ELISA. Type I collagen, fibronectin, laminin and Type IV collagen were measured for three independent extractions and presented as mean ± S.D. (B). Adhered human mesenchymal stem cells (hMSCs) were incubated with either labeled exosomes or free-dye for 24 h, as described in Materials and Methods section. Following incubation, the cells were washed with PBS, fixed with paraformaldehyde and fluorescence intensity was measured in a plate reader. Exosome uptake is presented as mean ± S.D. of fluorescence intensity of osteoblast (Os-Exo), adipocyte (Ad-Exo) and hMSC (hMSC-Exo). The hMSCs were supplemented with labeled exosomes for 2 h either at 37°C (solid bar) in the presence of 5µM (vertical lines bars) and 10µM (horizontal lines bars) of chlorpromazine. While incubation of hMSCs with exosomes at 4°C (slanted line bars) inhibited the uptake of exosomes. One-way ANOVA was used to evaluate statistical significance between groups (*p ≤ 0.005).

    Techniques Used: Microscopy, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Fluorescence

    Effect of extracellular matrix and exosomes on gene expression in human mesenchymal stem cell differentiation. Human mesenchymal stem cells (hMSCs) were differentiated in either tissue culture plate (TCP, open bars) or cell type specific extracellular matrix (ECM, vertical line bars). The hMSCs differentiated in TCP (TCP/Exo, horizontal line bars) and ECM (ECM/Exo, checkered bars) were further differentiated in the presence of either osteoblast exosomes (A) or adipocyte exosomes (B). Relative gene expressions presented represent fold changes with respect to undifferentiated hMSCs. Osteogenic (OC, RUNX2, OSX and OPN) and adipogenic (C/EBPα, LPL, ADPN and PPARγ) genes were quantitated by RT-qPCR using gene specific primers. The OC (C) and ADPN (D) promoter activities were assessed during differentiation of hMSCs into osteoblast and adipocytes at different conditions, respectively. The hMSCs grown on either TCP or ECM were supplemented with exosomes as described in Materials and Methods section. The OC and ADPN promoter constructs were transfected individually into the hMSCs on day-15 of differentiation towards osteogenic and adipogenic lineage, respectively. Following 24 h transfection, luciferase activity was measured in the cell lysates. One-way ANOVA was used to evaluate statistical significance between groups (*p ≤ 0.005).
    Figure Legend Snippet: Effect of extracellular matrix and exosomes on gene expression in human mesenchymal stem cell differentiation. Human mesenchymal stem cells (hMSCs) were differentiated in either tissue culture plate (TCP, open bars) or cell type specific extracellular matrix (ECM, vertical line bars). The hMSCs differentiated in TCP (TCP/Exo, horizontal line bars) and ECM (ECM/Exo, checkered bars) were further differentiated in the presence of either osteoblast exosomes (A) or adipocyte exosomes (B). Relative gene expressions presented represent fold changes with respect to undifferentiated hMSCs. Osteogenic (OC, RUNX2, OSX and OPN) and adipogenic (C/EBPα, LPL, ADPN and PPARγ) genes were quantitated by RT-qPCR using gene specific primers. The OC (C) and ADPN (D) promoter activities were assessed during differentiation of hMSCs into osteoblast and adipocytes at different conditions, respectively. The hMSCs grown on either TCP or ECM were supplemented with exosomes as described in Materials and Methods section. The OC and ADPN promoter constructs were transfected individually into the hMSCs on day-15 of differentiation towards osteogenic and adipogenic lineage, respectively. Following 24 h transfection, luciferase activity was measured in the cell lysates. One-way ANOVA was used to evaluate statistical significance between groups (*p ≤ 0.005).

    Techniques Used: Expressing, Cell Differentiation, Quantitative RT-PCR, Construct, Transfection, Luciferase, Activity Assay

    Effect of cell specific exosomes on extracellular matrix directed human mesenchymal stem cell lineage. Human mesenchymal stem cells (hMSCs) were differentiated on either osteoblast (Os-ECM) or adipocyte (Ad-ECM) extracellular matrix (ECM). During osteogenic differentiation on Os-ECM the cells were supplemented with either osteoblast exosomes [Os-ECM/Os-Exo; (A)] or adipocyte exosomes [Os-ECM/Ad-Exo (B)], while during adipogenic differentiation on Ad-ECM the cells were supplemented with either osteoblast exosomes [Ad-ECM/Os-Exo (C)] or adipocyte exosomes [Ad-ECM/Ad-Exo (B)]. After 15 days of differentiation the osteogenic [OC, RUNX2 and OSX) and adipogenic (C/EBPα, ADPN and PPARγ) specific gene expressions were quantitated by RT-qPCR analyses using specific primers. The OC (E) and ADPN (F) promoter activities were assessed during the differentiation of hMSCs to osteoblast or adipocyte on either Os-ECM or Ad-ECM with respective exosomes. The promoter activities were also measured in the presence of 10 µM chlorpromazine supplemented with exosomes. The siRNAs targeting RUNX2 and PPARγ were transfected 8 h prior to supplementation of exosomes. One-way Anova was used to evaluate the statistical significance (*p ≤ 0.005).
    Figure Legend Snippet: Effect of cell specific exosomes on extracellular matrix directed human mesenchymal stem cell lineage. Human mesenchymal stem cells (hMSCs) were differentiated on either osteoblast (Os-ECM) or adipocyte (Ad-ECM) extracellular matrix (ECM). During osteogenic differentiation on Os-ECM the cells were supplemented with either osteoblast exosomes [Os-ECM/Os-Exo; (A)] or adipocyte exosomes [Os-ECM/Ad-Exo (B)], while during adipogenic differentiation on Ad-ECM the cells were supplemented with either osteoblast exosomes [Ad-ECM/Os-Exo (C)] or adipocyte exosomes [Ad-ECM/Ad-Exo (B)]. After 15 days of differentiation the osteogenic [OC, RUNX2 and OSX) and adipogenic (C/EBPα, ADPN and PPARγ) specific gene expressions were quantitated by RT-qPCR analyses using specific primers. The OC (E) and ADPN (F) promoter activities were assessed during the differentiation of hMSCs to osteoblast or adipocyte on either Os-ECM or Ad-ECM with respective exosomes. The promoter activities were also measured in the presence of 10 µM chlorpromazine supplemented with exosomes. The siRNAs targeting RUNX2 and PPARγ were transfected 8 h prior to supplementation of exosomes. One-way Anova was used to evaluate the statistical significance (*p ≤ 0.005).

    Techniques Used: Quantitative RT-PCR, Transfection

    9) Product Images from "Effect of silver nanoparticles on human mesenchymal stem cell differentiation"

    Article Title: Effect of silver nanoparticles on human mesenchymal stem cell differentiation

    Journal: Beilstein Journal of Nanotechnology

    doi: 10.3762/bjnano.5.214

    Influence of different Ag-NP/Ag + ion concentrations on the viability of undifferentiated hMSCs (A) and adipogenic-differentiated hMSCs (B). After 14 d of incubation, viable cells were stained with calcein-AM (green fluorescence) and quantified by using
    Figure Legend Snippet: Influence of different Ag-NP/Ag + ion concentrations on the viability of undifferentiated hMSCs (A) and adipogenic-differentiated hMSCs (B). After 14 d of incubation, viable cells were stained with calcein-AM (green fluorescence) and quantified by using

    Techniques Used: Incubation, Staining, Fluorescence

    10) Product Images from "Smurf2-mediated degradation of EZH2 enhances neuron differentiation and improves functional recovery after ischaemic stroke"

    Article Title: Smurf2-mediated degradation of EZH2 enhances neuron differentiation and improves functional recovery after ischaemic stroke

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201783

    Smurf2-mediated degradation of EZH2 induces neuron differentiation of hMSCs. In proliferating hMSCs, EZH2 binds to the PPARγ promoter to repress neuron differentiation. After induction to neuron differentiation, EZH2 dissociates from the PPARγ promoter and is downregulated through Smurf2-mediated degradation to enhance gene expression of PPARγ to promote hMSC neuron differentiation.
    Figure Legend Snippet: Smurf2-mediated degradation of EZH2 induces neuron differentiation of hMSCs. In proliferating hMSCs, EZH2 binds to the PPARγ promoter to repress neuron differentiation. After induction to neuron differentiation, EZH2 dissociates from the PPARγ promoter and is downregulated through Smurf2-mediated degradation to enhance gene expression of PPARγ to promote hMSC neuron differentiation.

    Techniques Used: Expressing

    Downregulation of EZH2 promotes neuron differentiation of hMSCs. A.Representative undifferentiated primary hMSCs (top left) and differentiated neuron with cell body morphologies (black arrow) as well as extended neurite-like structures (bottom left), which shows MAP2 expression (white arrow) by immunocytochemical analysis (right). Scale bars: 50 µm. B. Lentiviral-mediated shRNA interference targeting EZH2 was used to allow for the generation of hMSCs stably expressing shEZH2 (shEZH2 #C and #D). C. The effects of 3A6-hMSCs and primary hMSCs stably expressing shRNA targeting EZH2 (shEZH2 #C) and treat with NIM for indicated time intervals during neuron differentiation (Diff). The total cell lysate at each time interval was extracted and immunoblotted with the indicated antibodies. The plots (bottom) represent the relative density of EZH2, MSC marker (CD105), and neuron marker (MAP2) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). Source data is available for this figure in the Supporting Information. D. Cell morphology of 3A6-hMSCs with or without stably expressing shEZH2 (shEZH2 #C) and treat with NIM at indicated time interval during neuron differentiation was observed under an inverted phase microscope. The inset is an enlarged image of the boxed (white) region. Scale bars: 50 µm. E. Quantitative PCR analysis of EZH2, MAP2 (neuron marker), troponin T (TnT; cardiac marker), osteopontin (OPN; osteogenic marker) mRNA levels in NIM-treated 3A6-hMSCs at indicated time intervals during neuron differentiation (Diff). A change in the neuron marker (MAP2) was observed. Error bars represent the SEM from three independent experiments. Significant p values are indicated ( n = 3, Student's t -test).
    Figure Legend Snippet: Downregulation of EZH2 promotes neuron differentiation of hMSCs. A.Representative undifferentiated primary hMSCs (top left) and differentiated neuron with cell body morphologies (black arrow) as well as extended neurite-like structures (bottom left), which shows MAP2 expression (white arrow) by immunocytochemical analysis (right). Scale bars: 50 µm. B. Lentiviral-mediated shRNA interference targeting EZH2 was used to allow for the generation of hMSCs stably expressing shEZH2 (shEZH2 #C and #D). C. The effects of 3A6-hMSCs and primary hMSCs stably expressing shRNA targeting EZH2 (shEZH2 #C) and treat with NIM for indicated time intervals during neuron differentiation (Diff). The total cell lysate at each time interval was extracted and immunoblotted with the indicated antibodies. The plots (bottom) represent the relative density of EZH2, MSC marker (CD105), and neuron marker (MAP2) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). Source data is available for this figure in the Supporting Information. D. Cell morphology of 3A6-hMSCs with or without stably expressing shEZH2 (shEZH2 #C) and treat with NIM at indicated time interval during neuron differentiation was observed under an inverted phase microscope. The inset is an enlarged image of the boxed (white) region. Scale bars: 50 µm. E. Quantitative PCR analysis of EZH2, MAP2 (neuron marker), troponin T (TnT; cardiac marker), osteopontin (OPN; osteogenic marker) mRNA levels in NIM-treated 3A6-hMSCs at indicated time intervals during neuron differentiation (Diff). A change in the neuron marker (MAP2) was observed. Error bars represent the SEM from three independent experiments. Significant p values are indicated ( n = 3, Student's t -test).

    Techniques Used: Expressing, shRNA, Stable Transfection, Marker, Microscopy, Real-time Polymerase Chain Reaction

    K421 of EZH2 is critical for Smurf2-mediated EZH2 degradation, which is required for neuron differentiation. Source data is available for this figure in the Supporting Information. A. HEK 293 cells were cotransfected wild-type or K to R mutants of Myc-EZH2 with HA-Ub with or without Flag-Smurf2 plasmids for 24 h. Total cell lysate was extracted, and expression of exogenous Myc-EZH2 and Flag-Smurf2 was examined (immunoblot, top). The plot (bottom) represents the relative density of EZH2 (wild-type and mutants) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). B. Expression of ectopic Myc-EZH2 (wild-type or K421R) in the absence or presence of Flag-Smurf2 (wild-type or C716A) with or without MG132 in HEK 293 cells. C. HEK 293 cells were transfected with Myc-EZH2 (wild-type or K421R) plasmids. Each transfectant was treated with 50 µM cycloheximide for the indicated times. The levels of indicated proteins were determined by immunoblotting (top). The percentage of EZH2 degradation was calculated by the relative level of Myc-EZH2 (WT: red and K421R: blue) normalized to α-tubulin (bottom). Error bars represent the SEM from three independent experiments ( n = 3). D. Immunoblotting of Smurf2 protein levels of 3A6-hMSCs with Smurf2 knockdown (shSmurf2 #E and #F). E. 3A6-hMSCs stably expressing Smurf2 shRNA (shSmurf2 #E) or pretreated with MG132 were treated with NIM for 5 days. EZH2 was then immunoprecipitated by an EZH2 antibody and blotted with an anti-ubiquitin antibody. F. The total cell lysates with or without induction of neuron differentiation in 3A6-hMSCs for 5 days and cells stably expressing Smurf2 shRNA (shSmurf2 #E) at the neuron stage were immunoblotted with the indicated antibodies. Changes in the MSC (CD105) and the neuron (MAP2) markers were analysed by immunoblotting. G. Cell morphology of NIM-treated 3A6-hMSCs with or without stably expressing shSmurf2 (shSmurf2 #E) at indicated times during neuron differentiation was observed under an inverted phase microscope. The inset is an enlarged image of the boxed (white) region. Scale bars: 50 µm.
    Figure Legend Snippet: K421 of EZH2 is critical for Smurf2-mediated EZH2 degradation, which is required for neuron differentiation. Source data is available for this figure in the Supporting Information. A. HEK 293 cells were cotransfected wild-type or K to R mutants of Myc-EZH2 with HA-Ub with or without Flag-Smurf2 plasmids for 24 h. Total cell lysate was extracted, and expression of exogenous Myc-EZH2 and Flag-Smurf2 was examined (immunoblot, top). The plot (bottom) represents the relative density of EZH2 (wild-type and mutants) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). B. Expression of ectopic Myc-EZH2 (wild-type or K421R) in the absence or presence of Flag-Smurf2 (wild-type or C716A) with or without MG132 in HEK 293 cells. C. HEK 293 cells were transfected with Myc-EZH2 (wild-type or K421R) plasmids. Each transfectant was treated with 50 µM cycloheximide for the indicated times. The levels of indicated proteins were determined by immunoblotting (top). The percentage of EZH2 degradation was calculated by the relative level of Myc-EZH2 (WT: red and K421R: blue) normalized to α-tubulin (bottom). Error bars represent the SEM from three independent experiments ( n = 3). D. Immunoblotting of Smurf2 protein levels of 3A6-hMSCs with Smurf2 knockdown (shSmurf2 #E and #F). E. 3A6-hMSCs stably expressing Smurf2 shRNA (shSmurf2 #E) or pretreated with MG132 were treated with NIM for 5 days. EZH2 was then immunoprecipitated by an EZH2 antibody and blotted with an anti-ubiquitin antibody. F. The total cell lysates with or without induction of neuron differentiation in 3A6-hMSCs for 5 days and cells stably expressing Smurf2 shRNA (shSmurf2 #E) at the neuron stage were immunoblotted with the indicated antibodies. Changes in the MSC (CD105) and the neuron (MAP2) markers were analysed by immunoblotting. G. Cell morphology of NIM-treated 3A6-hMSCs with or without stably expressing shSmurf2 (shSmurf2 #E) at indicated times during neuron differentiation was observed under an inverted phase microscope. The inset is an enlarged image of the boxed (white) region. Scale bars: 50 µm.

    Techniques Used: Expressing, Transfection, Stable Transfection, shRNA, Immunoprecipitation, Microscopy

    Degradation of EZH2 via the ubiquitin-proteasome-dependent degradation pathway requires Smurf2. Source data is available for this figure in the Supporting Information. A. 3A6-hMSCs were treated with or without NIM and/or proteasome inhibitor MG132 (5 µM) for 5 days. EZH2 was then immunoprecipitated by an EZH2 antibody and blotted with an anti-ubiquitin antibody (immunoblot, top). The plots (bottom) represent the relative density of EZH2 determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). B. RT-PCR analysis of Smurf2 mRNA expression during neuron differentiation on Day 1 of 3A6-hMSCs (RT-PCR, top). The plot (bottom) represents the relative density of Smurf2, determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). C. Immunoblotting analysis of Smurf2 protein levels during neuron differentiation on Day 1 of both 3A6 and primary hMSCs. D. Ectopic expression of Flag-Smurf2 and Myc-EZH2 in HEK 293 cells with or without MG132 and co-immunoprecipitation of Smurf2 and EZH2. E. Co-immunoprecipitation of endogenous Smurf2 and EZH2 in 3A6-hMSCs treated with NIM for 1 day with or without MG132. F. In vivo ubiquitination assay. Ectopic expression of HA-Ubiquitin (Ub), Myc-EZH2, Flag-Smurf2, or Flag-Smurf2-C716A (catalytically inactive mutant of Smurf2) in HEK 293 cells with or without MG132. Polyubiquitinated EZH2 was detected by immunoprecipitation of HA-tagged ubiquitin followed by immunoblotting for Myc-EZH2.
    Figure Legend Snippet: Degradation of EZH2 via the ubiquitin-proteasome-dependent degradation pathway requires Smurf2. Source data is available for this figure in the Supporting Information. A. 3A6-hMSCs were treated with or without NIM and/or proteasome inhibitor MG132 (5 µM) for 5 days. EZH2 was then immunoprecipitated by an EZH2 antibody and blotted with an anti-ubiquitin antibody (immunoblot, top). The plots (bottom) represent the relative density of EZH2 determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). B. RT-PCR analysis of Smurf2 mRNA expression during neuron differentiation on Day 1 of 3A6-hMSCs (RT-PCR, top). The plot (bottom) represents the relative density of Smurf2, determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). C. Immunoblotting analysis of Smurf2 protein levels during neuron differentiation on Day 1 of both 3A6 and primary hMSCs. D. Ectopic expression of Flag-Smurf2 and Myc-EZH2 in HEK 293 cells with or without MG132 and co-immunoprecipitation of Smurf2 and EZH2. E. Co-immunoprecipitation of endogenous Smurf2 and EZH2 in 3A6-hMSCs treated with NIM for 1 day with or without MG132. F. In vivo ubiquitination assay. Ectopic expression of HA-Ubiquitin (Ub), Myc-EZH2, Flag-Smurf2, or Flag-Smurf2-C716A (catalytically inactive mutant of Smurf2) in HEK 293 cells with or without MG132. Polyubiquitinated EZH2 was detected by immunoprecipitation of HA-tagged ubiquitin followed by immunoblotting for Myc-EZH2.

    Techniques Used: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, In Vivo, Ubiquitin Assay, Mutagenesis

    11) Product Images from "Hypoxic mesenchymal stem cells ameliorate acute kidney ischemia-reperfusion injury via enhancing renal tubular autophagy"

    Article Title: Hypoxic mesenchymal stem cells ameliorate acute kidney ischemia-reperfusion injury via enhancing renal tubular autophagy

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02374-x

    Hypoxic rat mesenchymal stem cells (HMSCs) ameliorated hypoxia-reoxygenation (H/R) injured renal tubular cells through enhancing autophagy. a H/R injury (hypoxia for 24 h followed by reoxygenation for 6 h) significantly decreased cell survival in NRK-52E cells determined by an MTT assay. Either co-culture with HMSCs or addition of HMSC-conditioned medium (CM) decreased H/R-injury-induced cell death. Addition of 3-methyl adenine (3-MA), an autophagy inhibitor, abolished the protective effects of HSMCs and HMSC-CM. * p
    Figure Legend Snippet: Hypoxic rat mesenchymal stem cells (HMSCs) ameliorated hypoxia-reoxygenation (H/R) injured renal tubular cells through enhancing autophagy. a H/R injury (hypoxia for 24 h followed by reoxygenation for 6 h) significantly decreased cell survival in NRK-52E cells determined by an MTT assay. Either co-culture with HMSCs or addition of HMSC-conditioned medium (CM) decreased H/R-injury-induced cell death. Addition of 3-methyl adenine (3-MA), an autophagy inhibitor, abolished the protective effects of HSMCs and HMSC-CM. * p

    Techniques Used: MTT Assay, Co-Culture Assay

    Effect of hypoxic rat mesenchymal stem cells (HMSCs) on oxidative stress induced by renal ischemia-reperfusion (I/R) injury. a In vivo detection of reactive oxygen species (ROS) in the rat kidneys subjected to sham-operation (Sham), ischemia-reperfusion surgery followed by intra-renal arterial injection (IA) of either phosphate-buffered saline (I/R+PBS), normoxia-cultured mesenchymal stem cells (I/R+MSC, 5 × 10 5 cells per rat), or HMSCs (I/R+HMSC, 5 × 10 5 cells per rat) was measured immediately after reperfusion. The amount of ROS was expressed as chemiluminescence counts/10 s. Please see the “Material and methods” for details. b Quantification of in vivo detection of the reactive oxygen species in the rat kidneys of Sham, I/R+PBS, I/R+MSC, and I/R+HMSC groups. ** p
    Figure Legend Snippet: Effect of hypoxic rat mesenchymal stem cells (HMSCs) on oxidative stress induced by renal ischemia-reperfusion (I/R) injury. a In vivo detection of reactive oxygen species (ROS) in the rat kidneys subjected to sham-operation (Sham), ischemia-reperfusion surgery followed by intra-renal arterial injection (IA) of either phosphate-buffered saline (I/R+PBS), normoxia-cultured mesenchymal stem cells (I/R+MSC, 5 × 10 5 cells per rat), or HMSCs (I/R+HMSC, 5 × 10 5 cells per rat) was measured immediately after reperfusion. The amount of ROS was expressed as chemiluminescence counts/10 s. Please see the “Material and methods” for details. b Quantification of in vivo detection of the reactive oxygen species in the rat kidneys of Sham, I/R+PBS, I/R+MSC, and I/R+HMSC groups. ** p

    Techniques Used: In Vivo, Injection, Cell Culture

    Hypoxic rat mesenchymal stem cells (HMSCs) decreased oxidative damage and increased anti-oxidant response in the ischemia-reperfusion (I/R)-injured rat kidneys. a , c Immunofluorescence staining for superoxide by dihydroethidium (DHE) staining and macrophage by anti-CD68 staining in renal tissues of the sham-operated rats (Sham), I/R-injured rats with PBS administration (PBS), I/R-injured rats with intra-renal arterial administration of HMSCs (IA-HMSCs), I/R-injured rats with intraperitoneal administration of HMSCs (IP-HMSCs), and I/R-injured rats with intraperitoneal administration of 100-fold concentrated HMSCs-conditioned medium (HMSC-CM). DHE staining was predominantly at the renal tubular cells and at some macrophages (arrowheads). 4′,6-Diamidino-2-phenylindole (DAPI) represented nuclear staining. Scale bar = 50 μm. ** p
    Figure Legend Snippet: Hypoxic rat mesenchymal stem cells (HMSCs) decreased oxidative damage and increased anti-oxidant response in the ischemia-reperfusion (I/R)-injured rat kidneys. a , c Immunofluorescence staining for superoxide by dihydroethidium (DHE) staining and macrophage by anti-CD68 staining in renal tissues of the sham-operated rats (Sham), I/R-injured rats with PBS administration (PBS), I/R-injured rats with intra-renal arterial administration of HMSCs (IA-HMSCs), I/R-injured rats with intraperitoneal administration of HMSCs (IP-HMSCs), and I/R-injured rats with intraperitoneal administration of 100-fold concentrated HMSCs-conditioned medium (HMSC-CM). DHE staining was predominantly at the renal tubular cells and at some macrophages (arrowheads). 4′,6-Diamidino-2-phenylindole (DAPI) represented nuclear staining. Scale bar = 50 μm. ** p

    Techniques Used: Immunofluorescence, Staining

    12) Product Images from "Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro"

    Article Title: Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064389

    A dose-dependent expression of humanized recombinant green fluorescent protein (hrGFP) gene. HeLa cells and human bone marrow-derived mesenchymal stem cells (hMSCs) were co-transduced with binary Tet-On lentivirus vectors harboring both the human glial cell line-derived neurotrophic factor (hGDNF) and hrGFP genes. The virus vector-containing medium was replaced with the medium in the presence (On) or absence (Off) of doxycycline (Dox) 8 hour after transduction. Serial doses of Dox ranging from 10 −4 – 10 4 ng/ml were tested to induce transgene expression. The cells were harvested and mean fluorescence intensity (MFI) units for hrGFP were examined by flow cytometry (FCM) 4 days after Dox treatment. The expression of hrGFP transgene in HeLa cells (A) and hMSCs (B) was regulated in a clear Dox dose-dependent manner.
    Figure Legend Snippet: A dose-dependent expression of humanized recombinant green fluorescent protein (hrGFP) gene. HeLa cells and human bone marrow-derived mesenchymal stem cells (hMSCs) were co-transduced with binary Tet-On lentivirus vectors harboring both the human glial cell line-derived neurotrophic factor (hGDNF) and hrGFP genes. The virus vector-containing medium was replaced with the medium in the presence (On) or absence (Off) of doxycycline (Dox) 8 hour after transduction. Serial doses of Dox ranging from 10 −4 – 10 4 ng/ml were tested to induce transgene expression. The cells were harvested and mean fluorescence intensity (MFI) units for hrGFP were examined by flow cytometry (FCM) 4 days after Dox treatment. The expression of hrGFP transgene in HeLa cells (A) and hMSCs (B) was regulated in a clear Dox dose-dependent manner.

    Techniques Used: Expressing, Recombinant, Derivative Assay, Transduction, Plasmid Preparation, Fluorescence, Flow Cytometry, Cytometry

    13) Product Images from "Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis"

    Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22855-z

    Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.
    Figure Legend Snippet: Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.

    Techniques Used: Cell Culture, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Functional Assay, Significance Assay, Binding Assay, ALP Assay, Activity Assay

    Analysis of preA-hMSCs-CM reveals altered secretion of proteins related to extracellular matrix, cell adhesion, angiogenesis and wound healing. ( A ) Schematic overview of hMSCs treatment to induce prelamin A accumulation. From each hMSCs line hMSCs accumulating prelamin A (preA-hMSCs) and control hMSCs (ctrl-hMSCs) were obtained in parallel. Prelamin A accumulation at the nuclear envelope was confirmed by confocal microscopy (red: prelamin A, blue: DAPI). Scale bar = 10 µm. Conditioned media from preA-hMSCs and ctrl-hMSCs were collected and subjected to proteomic analysis. Two independent hMSCs lines were use to obtain conditioned media in the case of antibody arrays (n = 2) and 4 independent hMSCs lines in the case of LC-MS (n = 4). ( B ) The differentially secreted proteins by preA-hMSCs (detected by antibody arrays and LC-MS) were interrogated in terms of functional annotation by DAVID Bioinformatics Resources. The representative Gene Ontology terms, grouped in clusters with an enrichment score of 1.5 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. ( C ) Real-time quantitative PCR was used to assess the expression of Runx2 in pre-hMSCs and ctrl-hMSCs. Runx2 mRNA expression was normalized to the control gene Gapdh and fold induction was then calculated in reference to ctrl-hMSCs. Results are expressed as mean ± SEM (n = 4).
    Figure Legend Snippet: Analysis of preA-hMSCs-CM reveals altered secretion of proteins related to extracellular matrix, cell adhesion, angiogenesis and wound healing. ( A ) Schematic overview of hMSCs treatment to induce prelamin A accumulation. From each hMSCs line hMSCs accumulating prelamin A (preA-hMSCs) and control hMSCs (ctrl-hMSCs) were obtained in parallel. Prelamin A accumulation at the nuclear envelope was confirmed by confocal microscopy (red: prelamin A, blue: DAPI). Scale bar = 10 µm. Conditioned media from preA-hMSCs and ctrl-hMSCs were collected and subjected to proteomic analysis. Two independent hMSCs lines were use to obtain conditioned media in the case of antibody arrays (n = 2) and 4 independent hMSCs lines in the case of LC-MS (n = 4). ( B ) The differentially secreted proteins by preA-hMSCs (detected by antibody arrays and LC-MS) were interrogated in terms of functional annotation by DAVID Bioinformatics Resources. The representative Gene Ontology terms, grouped in clusters with an enrichment score of 1.5 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. ( C ) Real-time quantitative PCR was used to assess the expression of Runx2 in pre-hMSCs and ctrl-hMSCs. Runx2 mRNA expression was normalized to the control gene Gapdh and fold induction was then calculated in reference to ctrl-hMSCs. Results are expressed as mean ± SEM (n = 4).

    Techniques Used: Confocal Microscopy, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Significance Assay, Real-time Polymerase Chain Reaction, Expressing

    14) Product Images from "Downregulation of DAB2IP Promotes Mesenchymal-To-Neuroepithelial Transition and Neuronal Differentiation of Human Mesenchymal Stem Cells"

    Article Title: Downregulation of DAB2IP Promotes Mesenchymal-To-Neuroepithelial Transition and Neuronal Differentiation of Human Mesenchymal Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075884

    Correlation between the reduction of DAB2IP expression and hMSC proliferation. A. Immunofluorescence staining of Ki-67 (green) in shRNA-transfected 3A6-hMSCs. B. Quantification of Ki-67-positive shRNA-transfected 3A6-hMSCs. Bars represent mean ± SEM (** p
    Figure Legend Snippet: Correlation between the reduction of DAB2IP expression and hMSC proliferation. A. Immunofluorescence staining of Ki-67 (green) in shRNA-transfected 3A6-hMSCs. B. Quantification of Ki-67-positive shRNA-transfected 3A6-hMSCs. Bars represent mean ± SEM (** p

    Techniques Used: Expressing, Immunofluorescence, Staining, shRNA, Transfection

    Reduction of DAB2IP expression during the neuronal induction of hMSCs. A. DAB2IP protein expression was readily detected in most of the 3A6-hMSCs (left), whereas in the NIM cells, DAB2IP expression was significantly reduced (right). Images were acquired under the same exposure conditions. B and C. DAB2IP expression gradually decreased during the neuronal induction of 3A6-hMSCs and primary hMSCs at both the mRNA (B, RT-PCR) and protein (C, western blot) levels. β-actin served as an internal control. The bar graphs (bottom) represent the relative density of DAB2IP as determined by scanning densitometric tracings.
    Figure Legend Snippet: Reduction of DAB2IP expression during the neuronal induction of hMSCs. A. DAB2IP protein expression was readily detected in most of the 3A6-hMSCs (left), whereas in the NIM cells, DAB2IP expression was significantly reduced (right). Images were acquired under the same exposure conditions. B and C. DAB2IP expression gradually decreased during the neuronal induction of 3A6-hMSCs and primary hMSCs at both the mRNA (B, RT-PCR) and protein (C, western blot) levels. β-actin served as an internal control. The bar graphs (bottom) represent the relative density of DAB2IP as determined by scanning densitometric tracings.

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

    Effect of DAB2IP expression on neuronal differentiation of hMSCs. A. Immunofluorescence staining of Tuj-1 expression (green) in 3A6-hMSCs infected with lentivirus-containing shRNAs against luciferase (shLuc) or DAB2IP (sh57 and sh59). B. Quantification of data presented in A as a percentage of Tuj-1-positive cells relative to the total number of cells. Bars represent mean ± SEM (** p
    Figure Legend Snippet: Effect of DAB2IP expression on neuronal differentiation of hMSCs. A. Immunofluorescence staining of Tuj-1 expression (green) in 3A6-hMSCs infected with lentivirus-containing shRNAs against luciferase (shLuc) or DAB2IP (sh57 and sh59). B. Quantification of data presented in A as a percentage of Tuj-1-positive cells relative to the total number of cells. Bars represent mean ± SEM (** p

    Techniques Used: Expressing, Immunofluorescence, Staining, Infection, Luciferase

    Effect of GSK3β on mesenchymal-to-neuroepithelial transition of hMSCs. A. Western blot analysis of whole-cell lysates from naïve 3A6-hMSCs. IP: immunoprecipitation. B. Western blot analysis of DAB2IP and Ser9 phosphorylated GSK3β (pGSK3β) in sh57 and sh59 DAB2IP-KD 3A6-hMSCs compared with shLuc control cells. The bar graphs (bottom) represent the relative density of DAB2IP, and pGSK3β as determined by scanning densitometric tracings. C. Western blot analysis for the MSC marker CD105 and the neuronal progenitor cell marker Nestin in naïve or LiCl-pretreated 3A6-hMSCs cultured with or without NIM. β-actin served as an internal control. The bar graphs (bottom) represent the relative density of CD105, Nestin, DAB2IP, and pGSK3β as determined by scanning densitometric tracings. D. Flow cytometry of MAP2-positive DAB2IP-KD hMSCs. Bars represent mean ± SEM (** p
    Figure Legend Snippet: Effect of GSK3β on mesenchymal-to-neuroepithelial transition of hMSCs. A. Western blot analysis of whole-cell lysates from naïve 3A6-hMSCs. IP: immunoprecipitation. B. Western blot analysis of DAB2IP and Ser9 phosphorylated GSK3β (pGSK3β) in sh57 and sh59 DAB2IP-KD 3A6-hMSCs compared with shLuc control cells. The bar graphs (bottom) represent the relative density of DAB2IP, and pGSK3β as determined by scanning densitometric tracings. C. Western blot analysis for the MSC marker CD105 and the neuronal progenitor cell marker Nestin in naïve or LiCl-pretreated 3A6-hMSCs cultured with or without NIM. β-actin served as an internal control. The bar graphs (bottom) represent the relative density of CD105, Nestin, DAB2IP, and pGSK3β as determined by scanning densitometric tracings. D. Flow cytometry of MAP2-positive DAB2IP-KD hMSCs. Bars represent mean ± SEM (** p

    Techniques Used: Western Blot, Immunoprecipitation, Marker, Cell Culture, Flow Cytometry, Cytometry

    15) Product Images from "MR Signal Characteristics of Viable and Apoptotic Human Mesenchymal Stem Cells in MASI for Treatment of Osteoarthritis"

    Article Title: MR Signal Characteristics of Viable and Apoptotic Human Mesenchymal Stem Cells in MASI for Treatment of Osteoarthritis

    Journal: Investigative radiology

    doi: 10.1097/RLI.0b013e3181ed566c

    Coronal T2 weighted SE images (TR 4000 ms/TE 18.27 ms) of a patella specimen with implanted hMSCs in cartilage defects. A: MASI constructs with ferumoxides labeled hMSCs show a marked negative signal effect. Labeled viable cells (A1) show relatively less
    Figure Legend Snippet: Coronal T2 weighted SE images (TR 4000 ms/TE 18.27 ms) of a patella specimen with implanted hMSCs in cartilage defects. A: MASI constructs with ferumoxides labeled hMSCs show a marked negative signal effect. Labeled viable cells (A1) show relatively less

    Techniques Used: Mass Spectrometry, Construct, Labeling

    16) Product Images from "In Vivo Bioluminescent Tracking of Mesenchymal Stem Cells Within Large Hydrogel Constructs"

    Article Title: In Vivo Bioluminescent Tracking of Mesenchymal Stem Cells Within Large Hydrogel Constructs

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2013.0587

    Characterization of green fluorescent protein (GFP)/luciferase (Luc)-labeled human mesenchymal stem cells (hMSCs). (A) Labeled hMSCs retained a fusiform morphology and expressed GFP (green) as early as 24 h following lentiviral cotransduction
    Figure Legend Snippet: Characterization of green fluorescent protein (GFP)/luciferase (Luc)-labeled human mesenchymal stem cells (hMSCs). (A) Labeled hMSCs retained a fusiform morphology and expressed GFP (green) as early as 24 h following lentiviral cotransduction

    Techniques Used: Luciferase, Labeling

    17) Product Images from "Methodological aspects of MRI of transplanted superparamagnetic iron oxide-labeled mesenchymal stem cells in live rat brain"

    Article Title: Methodological aspects of MRI of transplanted superparamagnetic iron oxide-labeled mesenchymal stem cells in live rat brain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186717

    MR and confocal fluorescence microscopy images of rat brain after intracerebral and intra-arterial transplantation of hMSCs double-labeled with SPIO microparticles and PKH26. (A) MRI of rat brain (T2WI and SWI) immediately after intracerebral injection of 10 5 hMSCs double-labeled with MC03F SPIO microparticles and PKH26. Hypointense regions indicate the location of SPIO labeled cells or probably extracellular SPIO microspheres. (B) High-magnification confocal micrographs of the hMSCs injection site. The same rat brain as in fig A. White arrowhead points to a double-labeled hMSC (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nucleus stained with DAPI is blue). Single clusters of extracellular iron can also be visualized (white arrows). The scale bars represent 10 μm. (C) Confocal panoramic micrographs of the hMSCs injection site (the same rat brain as in fig A). Labeled hMSCs are located along the track of the injection needle and in corpus callosum. The scale bars represent 500 μm. (D) MRI of rat brain (T2WI and SWI) after intra-arterial transplantation of 10 5 hMSCs labeled with SPIO-microparticles and PKH26. The white arrows on the SWI picture indicate the location of SPIO labeled cells. (E) High-magnification confocal micrographs (the same rat brain as in fig D) of transplanted hMSCs. White arrowhead points at double-labeled cells (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nuclei stained with DAPI is blue). The scale bars represent 20 μm.
    Figure Legend Snippet: MR and confocal fluorescence microscopy images of rat brain after intracerebral and intra-arterial transplantation of hMSCs double-labeled with SPIO microparticles and PKH26. (A) MRI of rat brain (T2WI and SWI) immediately after intracerebral injection of 10 5 hMSCs double-labeled with MC03F SPIO microparticles and PKH26. Hypointense regions indicate the location of SPIO labeled cells or probably extracellular SPIO microspheres. (B) High-magnification confocal micrographs of the hMSCs injection site. The same rat brain as in fig A. White arrowhead points to a double-labeled hMSC (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nucleus stained with DAPI is blue). Single clusters of extracellular iron can also be visualized (white arrows). The scale bars represent 10 μm. (C) Confocal panoramic micrographs of the hMSCs injection site (the same rat brain as in fig A). Labeled hMSCs are located along the track of the injection needle and in corpus callosum. The scale bars represent 500 μm. (D) MRI of rat brain (T2WI and SWI) after intra-arterial transplantation of 10 5 hMSCs labeled with SPIO-microparticles and PKH26. The white arrows on the SWI picture indicate the location of SPIO labeled cells. (E) High-magnification confocal micrographs (the same rat brain as in fig D) of transplanted hMSCs. White arrowhead points at double-labeled cells (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nuclei stained with DAPI is blue). The scale bars represent 20 μm.

    Techniques Used: Fluorescence, Microscopy, Transplantation Assay, Labeling, Magnetic Resonance Imaging, Injection, Staining

    The efficacy of hMSCs labeling with MC03F microparticles and the effects of labeling on cell viability and proliferation. (A)-(C): fluorescent microscopy of a hMSC culture 24 hours after labeling with MC03F microparticles. (A) MC03F microparticles (Dragon Green fluorescence). (B) Cell nuclei (DAPI blue fluorescence). (C) A and B merged. (D) Transmitted light microscopy of unstained hMSC culture (the same area as in A-C) 24 hours after labeling with MC03F microparticles. SPIO microparticles are visualized in the cytoplasm as brown spots around clear nuclei. (E) Flow cytometry analysis of cells 24 hours after labeling. The solid line presents data for labeled hMSCs and the dotted line—for unlabeled, control hMSCs. X-axis shows fluorescence intensity and Y-axis—cell counts. The plot demonstrates that about 96% of the cells contained Dragon Green fluorescent microparticles. (F) Influence of the labeling with MC03F microparticles on hMSCs viability and proliferation. Optical density (Y axis) is proportional to lactate dehydrogenase (LDH) activity in the cells and, hence, to the number of living cells. The presented histograms show that the numbers of living cells were not significantly different in labeled and control cultures indicating the absence of negative effects associated with labeling on cell viability and proliferation. The scale bars on all microphotographs mark 100 μm.
    Figure Legend Snippet: The efficacy of hMSCs labeling with MC03F microparticles and the effects of labeling on cell viability and proliferation. (A)-(C): fluorescent microscopy of a hMSC culture 24 hours after labeling with MC03F microparticles. (A) MC03F microparticles (Dragon Green fluorescence). (B) Cell nuclei (DAPI blue fluorescence). (C) A and B merged. (D) Transmitted light microscopy of unstained hMSC culture (the same area as in A-C) 24 hours after labeling with MC03F microparticles. SPIO microparticles are visualized in the cytoplasm as brown spots around clear nuclei. (E) Flow cytometry analysis of cells 24 hours after labeling. The solid line presents data for labeled hMSCs and the dotted line—for unlabeled, control hMSCs. X-axis shows fluorescence intensity and Y-axis—cell counts. The plot demonstrates that about 96% of the cells contained Dragon Green fluorescent microparticles. (F) Influence of the labeling with MC03F microparticles on hMSCs viability and proliferation. Optical density (Y axis) is proportional to lactate dehydrogenase (LDH) activity in the cells and, hence, to the number of living cells. The presented histograms show that the numbers of living cells were not significantly different in labeled and control cultures indicating the absence of negative effects associated with labeling on cell viability and proliferation. The scale bars on all microphotographs mark 100 μm.

    Techniques Used: Labeling, Microscopy, Fluorescence, Light Microscopy, Flow Cytometry, Cytometry, Activity Assay

    MR and histological images of a rat brain taken after intra-arterial administration of 5x10 5 hMSCs labeled with MC03F microparticles via the right internal carotid artery. MRI was performed immediately after cell transplantation and euthanasia–immediately after MRI. Top panel row, left to right: T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI images. Rectangles indicate the area of labeled cells distribution. Bottom panel, left to right: conventional fluorescence microscopy (low and high magnification), bright-field microscopy of Perls’ Prussian Blue stained sections, confocal fluorescence microscopy (low and high magnification). The scale bars in panoramic views of coronal sections represent 500 μm and at high-magnification pictures they correspond to 50 μm.
    Figure Legend Snippet: MR and histological images of a rat brain taken after intra-arterial administration of 5x10 5 hMSCs labeled with MC03F microparticles via the right internal carotid artery. MRI was performed immediately after cell transplantation and euthanasia–immediately after MRI. Top panel row, left to right: T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI images. Rectangles indicate the area of labeled cells distribution. Bottom panel, left to right: conventional fluorescence microscopy (low and high magnification), bright-field microscopy of Perls’ Prussian Blue stained sections, confocal fluorescence microscopy (low and high magnification). The scale bars in panoramic views of coronal sections represent 500 μm and at high-magnification pictures they correspond to 50 μm.

    Techniques Used: Labeling, Magnetic Resonance Imaging, Transplantation Assay, Fluorescence, Microscopy, Staining

    Quantitative evaluation of MRI data. (A) The medians of the values of the ratio of minimum to mean signal calculated from data obtained using different pulse sequences after injection of 10 1 , 10 2 or 10 3 of SPIO-labeled hMSCs or saline into the rat striatum. Data for cell concentrations higher than 10 3 are not shown on the graph, because for them the minimum signal was zero for most MRI pulse sequences. Whiskers on the plot represent interquartile range. (B) Volumes of the hypointensity zones calculated from SWI data obtained after injection of varying quantities of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). (C) Values of the ratio of minimum to mean signal calculated from SWI data obtained after injection of 10 1 , 10 2 or 10 3 of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). Greek letters show statistical significance of differences between pulse sequences (A), or saline-injected and SPIO-labeled cell-transplanted rats (B, C): α –p
    Figure Legend Snippet: Quantitative evaluation of MRI data. (A) The medians of the values of the ratio of minimum to mean signal calculated from data obtained using different pulse sequences after injection of 10 1 , 10 2 or 10 3 of SPIO-labeled hMSCs or saline into the rat striatum. Data for cell concentrations higher than 10 3 are not shown on the graph, because for them the minimum signal was zero for most MRI pulse sequences. Whiskers on the plot represent interquartile range. (B) Volumes of the hypointensity zones calculated from SWI data obtained after injection of varying quantities of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). (C) Values of the ratio of minimum to mean signal calculated from SWI data obtained after injection of 10 1 , 10 2 or 10 3 of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). Greek letters show statistical significance of differences between pulse sequences (A), or saline-injected and SPIO-labeled cell-transplanted rats (B, C): α –p

    Techniques Used: Magnetic Resonance Imaging, Injection, Labeling, Whisker Assay

    SWI and high-magnification confocal fluorescence microscopy of normal rat brain immediately after intra-arterial injection of 5x10 5 hMSCs labeled with MC03F microparticles. SWI allows detection of small groups and even single hMSCs found in brain tissue close or around cerebral blood vessels. The scale bars represent 50 μm.
    Figure Legend Snippet: SWI and high-magnification confocal fluorescence microscopy of normal rat brain immediately after intra-arterial injection of 5x10 5 hMSCs labeled with MC03F microparticles. SWI allows detection of small groups and even single hMSCs found in brain tissue close or around cerebral blood vessels. The scale bars represent 50 μm.

    Techniques Used: Fluorescence, Microscopy, Injection, Labeling

    MR images of live rat brains after stereotaxic injection of 20 μl saline or different quantities (from 10 1 to 10 5 ) of SPIO-labeled hMSCs in 20 μl saline into the right striatum. The images were taken immediately after cell transplantation utilizing different MRI pulse sequences—T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI.
    Figure Legend Snippet: MR images of live rat brains after stereotaxic injection of 20 μl saline or different quantities (from 10 1 to 10 5 ) of SPIO-labeled hMSCs in 20 μl saline into the right striatum. The images were taken immediately after cell transplantation utilizing different MRI pulse sequences—T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI.

    Techniques Used: Injection, Labeling, Transplantation Assay, Magnetic Resonance Imaging

    MR and histological images of the sites of stereotaxic injection of 20 μl of saline or varying numbers of SPIO-labeled hMSCs suspended in 20 μl of saline into the right striatum of rats. Top panel row: T2WI. Second from top panel row: SWI. Middle panel row: conventional fluorescence microscopy. Cell nuclei stained with DAPI look blue. Transplanted hMSCs containing MC03F microparticles in their cytoplasm emit green fluorescence. Second from bottom panel row: confocal fluorescence microscopy. Bottom panel row: conventional microscopy of Perls’ Prussian Blue stained sections. The scale bars represent 500 μm in panoramic views of coronal sections and 50 μm in high-magnification images.
    Figure Legend Snippet: MR and histological images of the sites of stereotaxic injection of 20 μl of saline or varying numbers of SPIO-labeled hMSCs suspended in 20 μl of saline into the right striatum of rats. Top panel row: T2WI. Second from top panel row: SWI. Middle panel row: conventional fluorescence microscopy. Cell nuclei stained with DAPI look blue. Transplanted hMSCs containing MC03F microparticles in their cytoplasm emit green fluorescence. Second from bottom panel row: confocal fluorescence microscopy. Bottom panel row: conventional microscopy of Perls’ Prussian Blue stained sections. The scale bars represent 500 μm in panoramic views of coronal sections and 50 μm in high-magnification images.

    Techniques Used: Injection, Labeling, Fluorescence, Microscopy, Staining

    18) Product Images from "Photofunctionalization of Alginate Hydrogels to Promote Adhesion and Proliferation of Human Mesenchymal Stem Cells"

    Article Title: Photofunctionalization of Alginate Hydrogels to Promote Adhesion and Proliferation of Human Mesenchymal Stem Cells

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2012.0581

    (a) Fluorescence photomicrographs of live (FDA, green) and dead (EB, orange-red) encapsulated hMSCs cultured in vitro in the photocrosslinked ALG and ACR-RGD-ALG hydrogels after 14 and 28 days. The scale bar indicates 200 μm and all photographs were taken at the same magnification. (b) DNA content in the hMSC/hydrogel constructs normalized to dry weight.* p
    Figure Legend Snippet: (a) Fluorescence photomicrographs of live (FDA, green) and dead (EB, orange-red) encapsulated hMSCs cultured in vitro in the photocrosslinked ALG and ACR-RGD-ALG hydrogels after 14 and 28 days. The scale bar indicates 200 μm and all photographs were taken at the same magnification. (b) DNA content in the hMSC/hydrogel constructs normalized to dry weight.* p

    Techniques Used: Fluorescence, Cell Culture, In Vitro, Construct

    (a) Fluorescence photomicrographs of live (fluorescein diacetate [FDA], green) and dead (ethidium bromide [EB], orange-red) human mesenchymal stem cells (hMSCs) cultured on the surface of photocrosslinked ALG and ACR-RGD-ALG hydrogels for 5 days. (b) Fluorescence photomicrographs of 4′,6-diamidino-2-phenylindole (DAPI) positive stained hMSCs after 1, 2, and 5 days culture on the surface of photocrosslinked ALG and ACR-RGD-ALG hydrogels, and (c) quantification of adherent cell number. The scale bars indicate 200 μm and all photographs were taken at the same magnification. * p
    Figure Legend Snippet: (a) Fluorescence photomicrographs of live (fluorescein diacetate [FDA], green) and dead (ethidium bromide [EB], orange-red) human mesenchymal stem cells (hMSCs) cultured on the surface of photocrosslinked ALG and ACR-RGD-ALG hydrogels for 5 days. (b) Fluorescence photomicrographs of 4′,6-diamidino-2-phenylindole (DAPI) positive stained hMSCs after 1, 2, and 5 days culture on the surface of photocrosslinked ALG and ACR-RGD-ALG hydrogels, and (c) quantification of adherent cell number. The scale bars indicate 200 μm and all photographs were taken at the same magnification. * p

    Techniques Used: Fluorescence, Cell Culture, Staining

    19) Product Images from "Antimicrobial Properties of Mesenchymal Stem Cells: Therapeutic Potential for Cystic Fibrosis Infection, and Treatment"

    Article Title: Antimicrobial Properties of Mesenchymal Stem Cells: Therapeutic Potential for Cystic Fibrosis Infection, and Treatment

    Journal: Stem Cells International

    doi: 10.1155/2016/5303048

    Impact of blocking CFTR function on antimicrobial activity of MSCs. To mimic CF cells, healthy bone marrow derived hMSCs were cultured in the presence and absence of CFTR blocker I-172 (10 μ g/mL) without antibiotics for 24 hours. The hMSC supernatants were evaluated for the ability to impact Pseudomonas aeruginosa PA CFUs (a) and growth rate (b). Supernatants generated from CFTR deficient hMSCs were more inefficient at decreasing Pseudomonas aeruginosa CFUs ((b), P ≤ 0.05) and growth rate ((c), P ≤ 0.05) than hMSCs without CFTR activity blocked. Further, hMSCs with deficient CFTR activity had less ability to secrete LL-37 ((c), P ≤ 0.05) relative to controls. LL-37 production by bone marrow derived hMSCs is decreased when CFTR is blocked but can be increased by treating the cells with a variety of cytokine stimulators. hMSCs stimulated with cytokines IFN γ (100 ng/mL), IL-1B (50 ng/mL), and IL-12 (100 ng/mL) secreted significantly more LL-37 than unstimulated controls ((d), P ≤ 0.05, n = 4 different hMSC preparations).
    Figure Legend Snippet: Impact of blocking CFTR function on antimicrobial activity of MSCs. To mimic CF cells, healthy bone marrow derived hMSCs were cultured in the presence and absence of CFTR blocker I-172 (10 μ g/mL) without antibiotics for 24 hours. The hMSC supernatants were evaluated for the ability to impact Pseudomonas aeruginosa PA CFUs (a) and growth rate (b). Supernatants generated from CFTR deficient hMSCs were more inefficient at decreasing Pseudomonas aeruginosa CFUs ((b), P ≤ 0.05) and growth rate ((c), P ≤ 0.05) than hMSCs without CFTR activity blocked. Further, hMSCs with deficient CFTR activity had less ability to secrete LL-37 ((c), P ≤ 0.05) relative to controls. LL-37 production by bone marrow derived hMSCs is decreased when CFTR is blocked but can be increased by treating the cells with a variety of cytokine stimulators. hMSCs stimulated with cytokines IFN γ (100 ng/mL), IL-1B (50 ng/mL), and IL-12 (100 ng/mL) secreted significantly more LL-37 than unstimulated controls ((d), P ≤ 0.05, n = 4 different hMSC preparations).

    Techniques Used: Blocking Assay, Activity Assay, Derivative Assay, Cell Culture, Generated

    hMSCs in the PA (a) and SA (b) infection model: Cftr tm1Kth (CF) and wild type (WT) controls were infected with 10 5 CFUs of either Pseudomonas aeruginosa or Staphylococcus aureus impregnated into agarose beads to generate chronic gram negative or gram positive chronic infection models in CF. hMSCs were administered on day 1, 24 hours after infection. Mice were followed up to 10 days and were then euthanized for bacteria burden (BAL CFUs+ whole lung homogenate CFUs, n = 4 experiments with 10 animals in each group). hMSCs decreased bacteria burden ( P ≤ 0.05) in response to both pathogens.
    Figure Legend Snippet: hMSCs in the PA (a) and SA (b) infection model: Cftr tm1Kth (CF) and wild type (WT) controls were infected with 10 5 CFUs of either Pseudomonas aeruginosa or Staphylococcus aureus impregnated into agarose beads to generate chronic gram negative or gram positive chronic infection models in CF. hMSCs were administered on day 1, 24 hours after infection. Mice were followed up to 10 days and were then euthanized for bacteria burden (BAL CFUs+ whole lung homogenate CFUs, n = 4 experiments with 10 animals in each group). hMSCs decreased bacteria burden ( P ≤ 0.05) in response to both pathogens.

    Techniques Used: Infection, Mouse Assay

    20) Product Images from "Calcium Silicate/Chitosan-Coated Electrospun Poly (Lactic Acid) Fibers for Bone Tissue Engineering"

    Article Title: Calcium Silicate/Chitosan-Coated Electrospun Poly (Lactic Acid) Fibers for Bone Tissue Engineering

    Journal: Materials

    doi: 10.3390/ma10050501

    ( A ) COL; ( B ) alkaline phosphatase (ALP); ( C ) osteopontin (OPN); and ( D ) osteocalcin (OC) gene expression in the hMSCs were cultured on CS/CH-coated PLA mats for seven and 14 days. “*” indicates a significant difference ( p
    Figure Legend Snippet: ( A ) COL; ( B ) alkaline phosphatase (ALP); ( C ) osteopontin (OPN); and ( D ) osteocalcin (OC) gene expression in the hMSCs were cultured on CS/CH-coated PLA mats for seven and 14 days. “*” indicates a significant difference ( p

    Techniques Used: ALP Assay, Expressing, Cell Culture, Proximity Ligation Assay

    The immunofluorescence of hMSCs cultured on CS/CH-coated PLA mats for three and seven days.
    Figure Legend Snippet: The immunofluorescence of hMSCs cultured on CS/CH-coated PLA mats for three and seven days.

    Techniques Used: Immunofluorescence, Cell Culture, Proximity Ligation Assay

    ( A ) Adhesion and ( B ) proliferation of hMSCs cultured on CS/CH-coated PLA mats for different time points. “*” indicates a significant difference ( p
    Figure Legend Snippet: ( A ) Adhesion and ( B ) proliferation of hMSCs cultured on CS/CH-coated PLA mats for different time points. “*” indicates a significant difference ( p

    Techniques Used: Cell Culture, Proximity Ligation Assay

    SEM images of hMSCs adhered on CS/CH-coated PLA mats for 3 h and one day.
    Figure Legend Snippet: SEM images of hMSCs adhered on CS/CH-coated PLA mats for 3 h and one day.

    Techniques Used: Proximity Ligation Assay

    21) Product Images from "Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation"

    Article Title: Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

    Journal: Advanced Science

    doi: 10.1002/advs.201600347

    Surface curvature affects the F‐actin cytoskeleton and osteocalcin levels in hMSCs. Representative immunohistochemical images of osteocalcin in hMSCs on A) a concave and B) a convex spherical surface (κ = 1/175 µm −1 ) after 10 d in osteogenic medium (osteocalcin in green, nuclei in blue, and F‐actin in red). Scale bar 100 µm. Dashed lines highlight the contour of the spherical surface. C–D) Quantification of osteocalcin intensity/cell for different curvatures κ (mm −1 ) shown as mean values of all concave/convex surfaces (bar charts) and for the individual curvatures investigated (point charts). After 10 d in C) expansion medium and D) osteogenic medium, significant higher levels on convex spherical surfaces compared to flat and concave surfaces were revealed. E,F) Quantified F‐actin intensity/cell levels were highest on concave spherical surfaces after 10 d in E) expansion medium and F) osteogenic medium. Mean ± standard deviation. * P
    Figure Legend Snippet: Surface curvature affects the F‐actin cytoskeleton and osteocalcin levels in hMSCs. Representative immunohistochemical images of osteocalcin in hMSCs on A) a concave and B) a convex spherical surface (κ = 1/175 µm −1 ) after 10 d in osteogenic medium (osteocalcin in green, nuclei in blue, and F‐actin in red). Scale bar 100 µm. Dashed lines highlight the contour of the spherical surface. C–D) Quantification of osteocalcin intensity/cell for different curvatures κ (mm −1 ) shown as mean values of all concave/convex surfaces (bar charts) and for the individual curvatures investigated (point charts). After 10 d in C) expansion medium and D) osteogenic medium, significant higher levels on convex spherical surfaces compared to flat and concave surfaces were revealed. E,F) Quantified F‐actin intensity/cell levels were highest on concave spherical surfaces after 10 d in E) expansion medium and F) osteogenic medium. Mean ± standard deviation. * P

    Techniques Used: Immunohistochemistry, Standard Deviation

    22) Product Images from "EZH2 Regulates Neuronal Differentiation of Mesenchymal Stem Cells through PIP5K1C-dependent Calcium Signaling *"

    Article Title: EZH2 Regulates Neuronal Differentiation of Mesenchymal Stem Cells through PIP5K1C-dependent Calcium Signaling *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.185124

    The role of PIP5K1C in EZH2-mediated calcium signaling. A , undifferentiated and neuron-differentiated 3A6 hMSCs were collected and applied to ChIP assay with anti-EZH2 antibody or immunoglobulin (IgG, negative control). The immunoprecipitated chromatin
    Figure Legend Snippet: The role of PIP5K1C in EZH2-mediated calcium signaling. A , undifferentiated and neuron-differentiated 3A6 hMSCs were collected and applied to ChIP assay with anti-EZH2 antibody or immunoglobulin (IgG, negative control). The immunoprecipitated chromatin

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation

    A proposed model of EZH2-mediated PIP5K1C-dependent neuronal differentiation from hMSCs. In proliferating undifferentiated hMSCs, EZH2 protein binds to the promoter of PIP5K1C gene to repress its transcription to maintain the homeostasis of intracellular
    Figure Legend Snippet: A proposed model of EZH2-mediated PIP5K1C-dependent neuronal differentiation from hMSCs. In proliferating undifferentiated hMSCs, EZH2 protein binds to the promoter of PIP5K1C gene to repress its transcription to maintain the homeostasis of intracellular

    Techniques Used:

    Knockdown of EZH2 enhances neuronal differentiation from hMSCs in vitro and in vivo . A , mRNA expression profiles of neuron markers (NSE, PITX3, and NURR1), EZH2, PIP5K1C, as well as β-actin, were determined by RT-PCR at indicated time intervals
    Figure Legend Snippet: Knockdown of EZH2 enhances neuronal differentiation from hMSCs in vitro and in vivo . A , mRNA expression profiles of neuron markers (NSE, PITX3, and NURR1), EZH2, PIP5K1C, as well as β-actin, were determined by RT-PCR at indicated time intervals

    Techniques Used: In Vitro, In Vivo, Expressing, Reverse Transcription Polymerase Chain Reaction

    Negative regulation of IP 3 -mediated intracellular Ca 2+ contents by EZH2 in hMSCs. A , 3A6 hMSCs were infected without ( lane 1 ) or with lentivirus carrying shRNAs against luciferase ( lane 2 ) or EZH2 ( lane 3 ) gene. The shRNA against luciferase was used as
    Figure Legend Snippet: Negative regulation of IP 3 -mediated intracellular Ca 2+ contents by EZH2 in hMSCs. A , 3A6 hMSCs were infected without ( lane 1 ) or with lentivirus carrying shRNAs against luciferase ( lane 2 ) or EZH2 ( lane 3 ) gene. The shRNA against luciferase was used as

    Techniques Used: Infection, Luciferase, shRNA

    23) Product Images from "Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis"

    Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22855-z

    Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.
    Figure Legend Snippet: Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.

    Techniques Used: Cell Culture, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Functional Assay, Significance Assay, Binding Assay, ALP Assay, Activity Assay

    24) Product Images from "3D microniches reveal the importance of cell size and shape"

    Article Title: 3D microniches reveal the importance of cell size and shape

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02163-2

    F-actin filaments formation and polymerization in a 3D microniche. a Representative images of F-actin staining for hMSCs with different cell volumes and cell geometries after 24 h. b Quantification of the number of cells forming stress fibers in a 3D microniche with different sizes and geometries; n = 50–60 cells analyzed for each data point. c Immunofluorescence images of F-actin and G-actin for hMSCs with different volumes after 12 h. d Quantification of F- and G-actin levels 12 h after seeding in 3D microniches with different volumes. Total integrated fluorescence of phalloidin (F-actin) and DNaseI (G-actin) was normalized to the fluorescence of V 3 cells; n = 40–45 cells analyzed for each data point. e Immunofluorescence images of F-actin and G-actin for hMSCs with different geometries with V 3 volume after 12 h. f Comparison of normalized mean F- and G-actin intensity in cells with different shapes (cylinder and triangular prism) and aspect ratios (cubic and cuboid); n = 40–45 cells analyzed for each data point. g Left: F-actin staining for single hMSCs cultured in 3D microniches with different volumes. Representative cells were selected for each condition. Right: quantification of the number of cells forming stress fibers in 3D microniches with different volumes. Colored regions show cell volumes between 2000 ~ 3000, 3000 ~ 4000, 4000 ~ 5000, and > 5000 μm 3 , respectively. The values of cell volumes were presented on each image. Data are shown as mean ± s.d. for all panels, and * P
    Figure Legend Snippet: F-actin filaments formation and polymerization in a 3D microniche. a Representative images of F-actin staining for hMSCs with different cell volumes and cell geometries after 24 h. b Quantification of the number of cells forming stress fibers in a 3D microniche with different sizes and geometries; n = 50–60 cells analyzed for each data point. c Immunofluorescence images of F-actin and G-actin for hMSCs with different volumes after 12 h. d Quantification of F- and G-actin levels 12 h after seeding in 3D microniches with different volumes. Total integrated fluorescence of phalloidin (F-actin) and DNaseI (G-actin) was normalized to the fluorescence of V 3 cells; n = 40–45 cells analyzed for each data point. e Immunofluorescence images of F-actin and G-actin for hMSCs with different geometries with V 3 volume after 12 h. f Comparison of normalized mean F- and G-actin intensity in cells with different shapes (cylinder and triangular prism) and aspect ratios (cubic and cuboid); n = 40–45 cells analyzed for each data point. g Left: F-actin staining for single hMSCs cultured in 3D microniches with different volumes. Representative cells were selected for each condition. Right: quantification of the number of cells forming stress fibers in 3D microniches with different volumes. Colored regions show cell volumes between 2000 ~ 3000, 3000 ~ 4000, 4000 ~ 5000, and > 5000 μm 3 , respectively. The values of cell volumes were presented on each image. Data are shown as mean ± s.d. for all panels, and * P

    Techniques Used: Staining, Immunofluorescence, Fluorescence, Cell Culture

    Focal adhesions formation and cell tension in a 3D microniche. a Representative images of vinculin staining for single hMSCs cultured in 3D microniches with different volumes and geometries. b Fluorescent heat maps of ≥20 cells with the same volume ( V 3 ) but different geometries stained for vinculin. c Representative images of myosin IIa in cells of same geometry but different volumes. d Myosin IIa levels (per cell) as a function of cell volume. e Representative images of cells with different geometries but same volume ( V 3 ). f Myosin IIa levels as a function of cell shape (cylinder and triangular prism) or aspect ratio (cubic and cuboid). g Representative images of myosin IIa and F-actin before and after cells with V 3 treated with 50 μM Blebbistatin (Bleb); bar graph shows quantitation of the changes in the level of myosin IIa after treatment with 50 μM Blebbistatin (Bleb). Data are shown as mean ± s.d. for all panels; n = 45–60 cells analyzed for each data point and * P
    Figure Legend Snippet: Focal adhesions formation and cell tension in a 3D microniche. a Representative images of vinculin staining for single hMSCs cultured in 3D microniches with different volumes and geometries. b Fluorescent heat maps of ≥20 cells with the same volume ( V 3 ) but different geometries stained for vinculin. c Representative images of myosin IIa in cells of same geometry but different volumes. d Myosin IIa levels (per cell) as a function of cell volume. e Representative images of cells with different geometries but same volume ( V 3 ). f Myosin IIa levels as a function of cell shape (cylinder and triangular prism) or aspect ratio (cubic and cuboid). g Representative images of myosin IIa and F-actin before and after cells with V 3 treated with 50 μM Blebbistatin (Bleb); bar graph shows quantitation of the changes in the level of myosin IIa after treatment with 50 μM Blebbistatin (Bleb). Data are shown as mean ± s.d. for all panels; n = 45–60 cells analyzed for each data point and * P

    Techniques Used: Staining, Cell Culture, Quantitation Assay

    25) Product Images from "Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2"

    Article Title: Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2016.2694

    Nuclear factor-erythroid 2-related factor 2 (Nrf2) activation mediated by epigallocatechin-3-gallate (EGCG) pre-treatment suppresses H 2 O 2 -induced senescence and the expression of acetyl-p53 and p21 in human mesenchymal stem cells (hMSCs). (A) Senescence-associated β-galactosidase (SAβ-gal) staining analysis of control, H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed/Nrf2-siRNA-transfected and EGCG-pre-treated/H 2 O 2 -treated/control-siRNA-transfected cells. hMSCs were transiently transfected for 48 h with either Nrf2 or control siRNA and treated with 100 μ M EGCG for 6 h followed by H 2 O 2 exposure (200 μ M, 2 h). Twenty-four hours after H 2 O 2 exposure, the cells were subjected to SAβ-gal staining (blue cytoplasmic stain). Scale bar, 200 μ m. (B) Quantification of SAβ-gal activity. (C) Western blot analysis and quantification of Nrf2 at 48 h after Nrf2 siRNA or control siRNA transfection. (D–F) Western blot analysis and quantification of acetyl-p53 and p21 protein levels in each group. The levels determined in four independent experiments are presented as the means ± SEM. * P
    Figure Legend Snippet: Nuclear factor-erythroid 2-related factor 2 (Nrf2) activation mediated by epigallocatechin-3-gallate (EGCG) pre-treatment suppresses H 2 O 2 -induced senescence and the expression of acetyl-p53 and p21 in human mesenchymal stem cells (hMSCs). (A) Senescence-associated β-galactosidase (SAβ-gal) staining analysis of control, H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed/Nrf2-siRNA-transfected and EGCG-pre-treated/H 2 O 2 -treated/control-siRNA-transfected cells. hMSCs were transiently transfected for 48 h with either Nrf2 or control siRNA and treated with 100 μ M EGCG for 6 h followed by H 2 O 2 exposure (200 μ M, 2 h). Twenty-four hours after H 2 O 2 exposure, the cells were subjected to SAβ-gal staining (blue cytoplasmic stain). Scale bar, 200 μ m. (B) Quantification of SAβ-gal activity. (C) Western blot analysis and quantification of Nrf2 at 48 h after Nrf2 siRNA or control siRNA transfection. (D–F) Western blot analysis and quantification of acetyl-p53 and p21 protein levels in each group. The levels determined in four independent experiments are presented as the means ± SEM. * P

    Techniques Used: Activation Assay, Expressing, Staining, Transfection, Activity Assay, Western Blot

    Epigallocatechin-3-gallate (EGCG) pre-treatment reduces cellular senescence in H 2 O 2 -treated human mesenchymal stem cells (hMSCs). (A–D) Senescence-associated β-galactosidase (SAβ-gal) staining of control (Con) and hMSCs before and after H 2 O 2 exposure. hMSCs were treated with 50 or 100 μ M of EGCG for 6 h and then exposed to H 2 O 2 (200 μ M) for 2 h. Twenty-four hours after H 2 O 2 exposure, the cells were stained with SAβ-gal (blue cytoplasmic stain). Scale bar, 200 μ m. (E) Quantification of SAβ-gal activity. (F) Cell viability of hMSCs. MTT assays were performed 24 h after H 2 O 2 exposure. Changes in cell survival observed in three independent experiments are presented as the means ± SEM. * P
    Figure Legend Snippet: Epigallocatechin-3-gallate (EGCG) pre-treatment reduces cellular senescence in H 2 O 2 -treated human mesenchymal stem cells (hMSCs). (A–D) Senescence-associated β-galactosidase (SAβ-gal) staining of control (Con) and hMSCs before and after H 2 O 2 exposure. hMSCs were treated with 50 or 100 μ M of EGCG for 6 h and then exposed to H 2 O 2 (200 μ M) for 2 h. Twenty-four hours after H 2 O 2 exposure, the cells were stained with SAβ-gal (blue cytoplasmic stain). Scale bar, 200 μ m. (E) Quantification of SAβ-gal activity. (F) Cell viability of hMSCs. MTT assays were performed 24 h after H 2 O 2 exposure. Changes in cell survival observed in three independent experiments are presented as the means ± SEM. * P

    Techniques Used: Staining, Activity Assay, MTT Assay

    26) Product Images from "Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis"

    Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22855-z

    Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.
    Figure Legend Snippet: Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.

    Techniques Used: Cell Culture, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Functional Assay, Significance Assay, Binding Assay, ALP Assay, Activity Assay

    27) Product Images from "In Vivo Gene Activity of Human Mesenchymal Stem Cells After Scaffold-Mediated Local Transplantation"

    Article Title: In Vivo Gene Activity of Human Mesenchymal Stem Cells After Scaffold-Mediated Local Transplantation

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2013.0507

    Gene expression from hMSCs and resident cells in an immune-deficient model. hMSCs#1 were transplanted into immune-deficient Balb/c mice using the same strategy as was used for Group 1, where hMSCs were directly implanted without precultivation in vitro
    Figure Legend Snippet: Gene expression from hMSCs and resident cells in an immune-deficient model. hMSCs#1 were transplanted into immune-deficient Balb/c mice using the same strategy as was used for Group 1, where hMSCs were directly implanted without precultivation in vitro

    Techniques Used: Expressing, Mouse Assay, In Vitro

    28) Product Images from "Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier: An ex vivo study"

    Article Title: Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier: An ex vivo study

    Journal: Journal of Orthopaedic Translation

    doi: 10.1016/j.jot.2017.03.003

    Degenerative-loaded bovine intervertebral discs (IVDs) restored with human mesenchymal stem cells (hMSCs) in (A) and (B) fibrin (D) and (E) and saline solution following 7 days of dynamic culture. (A) and (D) Safranin O/Fast green stained transversal sections overviews (scale bar = 1 mm); (B) and (E) defect/tissue interface magnified views (scale bar = 100 μm); (C) combined phase contrast and fluorescent images of fibrin gel with PKH26-labelled hMSCs stained with calcein AM (yellow = viable hMSC, red = dead hMSC) and (D) nucleus pulposus tissue stained with calcein AM/ethidium homodimer (scale bar = 100 μm) (green = viable disc cell; red/yellow = dead disc cell). Note that fibrin prevents tissue swelling into the nucleotomised space and limits proteoglycan loss, as attested by the stronger Safranin O stain (A vs. D); fibrin can fill irregularly shaped defects (B). Note the homogeneous distribution of viable hMSCs in fibrin (C) and a majority of viable NP cells inside degenerated tissue (F). f = fibrin gel; np = nucleus pulposus s = saline.
    Figure Legend Snippet: Degenerative-loaded bovine intervertebral discs (IVDs) restored with human mesenchymal stem cells (hMSCs) in (A) and (B) fibrin (D) and (E) and saline solution following 7 days of dynamic culture. (A) and (D) Safranin O/Fast green stained transversal sections overviews (scale bar = 1 mm); (B) and (E) defect/tissue interface magnified views (scale bar = 100 μm); (C) combined phase contrast and fluorescent images of fibrin gel with PKH26-labelled hMSCs stained with calcein AM (yellow = viable hMSC, red = dead hMSC) and (D) nucleus pulposus tissue stained with calcein AM/ethidium homodimer (scale bar = 100 μm) (green = viable disc cell; red/yellow = dead disc cell). Note that fibrin prevents tissue swelling into the nucleotomised space and limits proteoglycan loss, as attested by the stronger Safranin O stain (A vs. D); fibrin can fill irregularly shaped defects (B). Note the homogeneous distribution of viable hMSCs in fibrin (C) and a majority of viable NP cells inside degenerated tissue (F). f = fibrin gel; np = nucleus pulposus s = saline.

    Techniques Used: Staining

    29) Product Images from "Biochemical and physical signal gradients in hydrogels to control stem cell behavior"

    Article Title: Biochemical and physical signal gradients in hydrogels to control stem cell behavior

    Journal: Advanced materials (Deerfield Beach, Fla.)

    doi: 10.1002/adma.201302364

    Characterization of BMP-2 and TGF-β1 gradient alginate hydrogels and response of photoencapsulated hMSCs in growth factor gradient hydrogels. (A) Quantification of BMP-2 (closed circle) and TGF-β1 (closed square) content in each segment
    Figure Legend Snippet: Characterization of BMP-2 and TGF-β1 gradient alginate hydrogels and response of photoencapsulated hMSCs in growth factor gradient hydrogels. (A) Quantification of BMP-2 (closed circle) and TGF-β1 (closed square) content in each segment

    Techniques Used:

    Characterization of RGD gradient alginate hydrogels and response of photoencapsulated hMSCs in RGD gradient hydrogels. (A) Quantification of RGD via ninhydrin assay in each segment of the RGD gradient hydrogels. (B) Representative live (FDA, green) /
    Figure Legend Snippet: Characterization of RGD gradient alginate hydrogels and response of photoencapsulated hMSCs in RGD gradient hydrogels. (A) Quantification of RGD via ninhydrin assay in each segment of the RGD gradient hydrogels. (B) Representative live (FDA, green) /

    Techniques Used:

    Characterization of stiffness gradient alginate hydrogels and response of photoencapsulated hMSCs in stiffness gradient hydrogels. (A) Modulus of each segment of stiffness gradient hydrogels. (B) Representative live/dead photomicrographs of photoencapsulated
    Figure Legend Snippet: Characterization of stiffness gradient alginate hydrogels and response of photoencapsulated hMSCs in stiffness gradient hydrogels. (A) Modulus of each segment of stiffness gradient hydrogels. (B) Representative live/dead photomicrographs of photoencapsulated

    Techniques Used:

    30) Product Images from "Von willebrand factor increases endothelial cell adhesiveness for human mesenchymal stem cells by activating p38 mitogen-activated protein kinase"

    Article Title: Von willebrand factor increases endothelial cell adhesiveness for human mesenchymal stem cells by activating p38 mitogen-activated protein kinase

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt35

    Effect of vWF on adhesion of hMSCs to collagen I coated or tissue culture treated plastic plates . Adhesion of hMSCs to collagen I coated and tissue culture plates was measured before and after immobilization of vWF. Before the adhesion assay vWF was removed and plates were washed with HBSS. Asterisks mark statistically significant differences compared to collagen I coated plate (t-test, P -value
    Figure Legend Snippet: Effect of vWF on adhesion of hMSCs to collagen I coated or tissue culture treated plastic plates . Adhesion of hMSCs to collagen I coated and tissue culture plates was measured before and after immobilization of vWF. Before the adhesion assay vWF was removed and plates were washed with HBSS. Asterisks mark statistically significant differences compared to collagen I coated plate (t-test, P -value

    Techniques Used: Cell Adhesion Assay, T-Test

    VWF stimulates HUVEC adhesiveness for hMSCs . (a) Shows changes in HUVEC adhesiveness for hMSCs caused by treatment with 4 μg/ml vWF (black circle) or HBSS (white circle) for 0 to 9 hours. Asterisks mark time points where adhesion of hMSCs to HUVECs treated with vWF was different (t-test, P -value
    Figure Legend Snippet: VWF stimulates HUVEC adhesiveness for hMSCs . (a) Shows changes in HUVEC adhesiveness for hMSCs caused by treatment with 4 μg/ml vWF (black circle) or HBSS (white circle) for 0 to 9 hours. Asterisks mark time points where adhesion of hMSCs to HUVECs treated with vWF was different (t-test, P -value

    Techniques Used: T-Test

    31) Product Images from "SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer"

    Article Title: SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer

    Journal: Protein & Cell

    doi: 10.1007/s13238-020-00728-4

    3TC attenuates SIRT7-deficient hMSCs senescence by antagonizing LINE1’s effects . (A) RT-qPCR analysis of the relative LINE1 genomic DNA content in SIRT7 −/− hMSCs treated with vehicle or 3TC. Data are presented as the means ± SEM. n = 4. *, P
    Figure Legend Snippet: 3TC attenuates SIRT7-deficient hMSCs senescence by antagonizing LINE1’s effects . (A) RT-qPCR analysis of the relative LINE1 genomic DNA content in SIRT7 −/− hMSCs treated with vehicle or 3TC. Data are presented as the means ± SEM. n = 4. *, P

    Techniques Used: Quantitative RT-PCR

    32) Product Images from "cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo"

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0711190105

    db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P
    Figure Legend Snippet: db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P

    Techniques Used: In Vivo, Cell Culture, Mouse Assay, Derivative Assay

    PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P
    Figure Legend Snippet: PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P

    Techniques Used: Activation Assay, In Vitro, ALP Assay, Expressing

    Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.
    Figure Legend Snippet: Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.

    Techniques Used: Expressing

    db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P
    Figure Legend Snippet: db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    33) Product Images from "Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation"

    Article Title: Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

    Journal: Advanced Science

    doi: 10.1002/advs.201600347

    Surface curvature affects the F‐actin cytoskeleton and osteocalcin levels in hMSCs. Representative immunohistochemical images of osteocalcin in hMSCs on A) a concave and B) a convex spherical surface (κ = 1/175 µm −1 ) after 10 d in osteogenic medium (osteocalcin in green, nuclei in blue, and F‐actin in red). Scale bar 100 µm. Dashed lines highlight the contour of the spherical surface. C–D) Quantification of osteocalcin intensity/cell for different curvatures κ (mm −1 ) shown as mean values of all concave/convex surfaces (bar charts) and for the individual curvatures investigated (point charts). After 10 d in C) expansion medium and D) osteogenic medium, significant higher levels on convex spherical surfaces compared to flat and concave surfaces were revealed. E,F) Quantified F‐actin intensity/cell levels were highest on concave spherical surfaces after 10 d in E) expansion medium and F) osteogenic medium. Mean ± standard deviation. * P
    Figure Legend Snippet: Surface curvature affects the F‐actin cytoskeleton and osteocalcin levels in hMSCs. Representative immunohistochemical images of osteocalcin in hMSCs on A) a concave and B) a convex spherical surface (κ = 1/175 µm −1 ) after 10 d in osteogenic medium (osteocalcin in green, nuclei in blue, and F‐actin in red). Scale bar 100 µm. Dashed lines highlight the contour of the spherical surface. C–D) Quantification of osteocalcin intensity/cell for different curvatures κ (mm −1 ) shown as mean values of all concave/convex surfaces (bar charts) and for the individual curvatures investigated (point charts). After 10 d in C) expansion medium and D) osteogenic medium, significant higher levels on convex spherical surfaces compared to flat and concave surfaces were revealed. E,F) Quantified F‐actin intensity/cell levels were highest on concave spherical surfaces after 10 d in E) expansion medium and F) osteogenic medium. Mean ± standard deviation. * P

    Techniques Used: Immunohistochemistry, Standard Deviation

    34) Product Images from "Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation"

    Article Title: Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193059

    Internalisation of exosomes in hMSCs. Confocal micrographs of hMSCs incubated for 24h with A) PBS (negative control); B) Exo_P6; C) Exo_D3 and D) Exo_D21. PKH67-stained exosomes are detected mainly in the cytoplasm of some of the cells. The intensity varied between individual cells. No PKH67-stained material was found in the negative control. A1-D1, blue channel; A2-D2, green channel; A3-D3, transmission (TD) channel; A4-D4, merged channels. Blue, DAPI-stained nucleus; Green, PKH67-stained exosomes. Scale bar: 20 μm.
    Figure Legend Snippet: Internalisation of exosomes in hMSCs. Confocal micrographs of hMSCs incubated for 24h with A) PBS (negative control); B) Exo_P6; C) Exo_D3 and D) Exo_D21. PKH67-stained exosomes are detected mainly in the cytoplasm of some of the cells. The intensity varied between individual cells. No PKH67-stained material was found in the negative control. A1-D1, blue channel; A2-D2, green channel; A3-D3, transmission (TD) channel; A4-D4, merged channels. Blue, DAPI-stained nucleus; Green, PKH67-stained exosomes. Scale bar: 20 μm.

    Techniques Used: Incubation, Negative Control, Staining, Transmission Assay

    MicroRNA profiles of exosomes and hMSCs during expansion and osteogenic differentiation. (A) Altered microRNA profiles of exosomes and hMSCs during expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs and (B) Altered microRNA profiles of exosomes derived from the expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs. The heat map diagram shows the result of the two-way hierarchical clustering of microRNAs and samples. The clustering is performed on all samples and on the top 50 microRNAs with the highest standard deviation. The normalised (dCq) values have been used for the analysis. Each row represents one microRNA and each column represents one sample. The microRNA clustering tree is shown on the left. The colour scale shown at the bottom illustrates the relative expression level of a microRNA across all samples: red colour represents an expression level above the mean, green colour represents expression lower than the mean.
    Figure Legend Snippet: MicroRNA profiles of exosomes and hMSCs during expansion and osteogenic differentiation. (A) Altered microRNA profiles of exosomes and hMSCs during expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs and (B) Altered microRNA profiles of exosomes derived from the expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs. The heat map diagram shows the result of the two-way hierarchical clustering of microRNAs and samples. The clustering is performed on all samples and on the top 50 microRNAs with the highest standard deviation. The normalised (dCq) values have been used for the analysis. Each row represents one microRNA and each column represents one sample. The microRNA clustering tree is shown on the left. The colour scale shown at the bottom illustrates the relative expression level of a microRNA across all samples: red colour represents an expression level above the mean, green colour represents expression lower than the mean.

    Techniques Used: Derivative Assay, Standard Deviation, Expressing

    Evaluation of osteogenic differentiation of hMSCs after exosome treatment. (A) ALP activity 14 d after treatment with exosomes or the appropriate controls; (B) Quantification of calcium in ECM after 21 d; (C) Quantification of phosphate in ECM after 21 d. In (A-C), bars indicate mean values whereas error bars denote standard errors of the mean (SEM). Small letters a, b and c represent statistical significance when compared with NCtrl_D0, NCtrl_D14/D21 and Sexo_Ctrl respectively, based on Bonferroni-corrected p value
    Figure Legend Snippet: Evaluation of osteogenic differentiation of hMSCs after exosome treatment. (A) ALP activity 14 d after treatment with exosomes or the appropriate controls; (B) Quantification of calcium in ECM after 21 d; (C) Quantification of phosphate in ECM after 21 d. In (A-C), bars indicate mean values whereas error bars denote standard errors of the mean (SEM). Small letters a, b and c represent statistical significance when compared with NCtrl_D0, NCtrl_D14/D21 and Sexo_Ctrl respectively, based on Bonferroni-corrected p value

    Techniques Used: ALP Assay, Activity Assay

    35) Product Images from "Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis"

    Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22855-z

    Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.
    Figure Legend Snippet: Secretome analysis of preA-adipocytes differentiated from hMSCs. ( A ) Schematic overview of hMSCs cell culture, induction of prelamin A accumulation by TPV treatment, adipogenesis, obtaining CM from hMSCs-derived adipocytes, and subsequent secretome analysis by antibody arrays and LC-MS approaches. Before adipogenic differentiation, induction of prelamin A accumulation in hMSCs was confirmed by confocal microscopy, red: prelamin A, blue: DAPI. Scale bar = 10 µm. (B) Functional annotation clustering of differentially secreted proteins in CM from preA-adipocytes, determined using the DAVID bioinformatic tool. The representative GO terms, grouped in clusters with an enrichment score of 7 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. (C) Venn diagrams showing overlap of 27 proteins between differentially secreted proteins by preA-hMSCs and preA-adipocytes. Gene ontology analysis of these proteins revealed significant over-representation of categories related to extracellular matrix, collagen binding and cell adhesion. ( D ) Six days after osteogenic differentiation of normal hMSCs in the presence of preA-adipocytes-CM or ctrl-adipocytes-CM, ALP activity was assessed. Results are expressed in reference to ALP activity of hMSCs cultured under ctrl-adipocytes-CM and represent mean ± SD, n = 6.

    Techniques Used: Cell Culture, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Functional Assay, Significance Assay, Binding Assay, ALP Assay, Activity Assay

    Analysis of preA-hMSCs-CM reveals altered secretion of proteins related to extracellular matrix, cell adhesion, angiogenesis and wound healing. ( A ) Schematic overview of hMSCs treatment to induce prelamin A accumulation. From each hMSCs line hMSCs accumulating prelamin A (preA-hMSCs) and control hMSCs (ctrl-hMSCs) were obtained in parallel. Prelamin A accumulation at the nuclear envelope was confirmed by confocal microscopy (red: prelamin A, blue: DAPI). Scale bar = 10 µm. Conditioned media from preA-hMSCs and ctrl-hMSCs were collected and subjected to proteomic analysis. Two independent hMSCs lines were use to obtain conditioned media in the case of antibody arrays (n = 2) and 4 independent hMSCs lines in the case of LC-MS (n = 4). ( B ) The differentially secreted proteins by preA-hMSCs (detected by antibody arrays and LC-MS) were interrogated in terms of functional annotation by DAVID Bioinformatics Resources. The representative Gene Ontology terms, grouped in clusters with an enrichment score of 1.5 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. ( C ) Real-time quantitative PCR was used to assess the expression of Runx2 in pre-hMSCs and ctrl-hMSCs. Runx2 mRNA expression was normalized to the control gene Gapdh and fold induction was then calculated in reference to ctrl-hMSCs. Results are expressed as mean ± SEM (n = 4).
    Figure Legend Snippet: Analysis of preA-hMSCs-CM reveals altered secretion of proteins related to extracellular matrix, cell adhesion, angiogenesis and wound healing. ( A ) Schematic overview of hMSCs treatment to induce prelamin A accumulation. From each hMSCs line hMSCs accumulating prelamin A (preA-hMSCs) and control hMSCs (ctrl-hMSCs) were obtained in parallel. Prelamin A accumulation at the nuclear envelope was confirmed by confocal microscopy (red: prelamin A, blue: DAPI). Scale bar = 10 µm. Conditioned media from preA-hMSCs and ctrl-hMSCs were collected and subjected to proteomic analysis. Two independent hMSCs lines were use to obtain conditioned media in the case of antibody arrays (n = 2) and 4 independent hMSCs lines in the case of LC-MS (n = 4). ( B ) The differentially secreted proteins by preA-hMSCs (detected by antibody arrays and LC-MS) were interrogated in terms of functional annotation by DAVID Bioinformatics Resources. The representative Gene Ontology terms, grouped in clusters with an enrichment score of 1.5 or above are presented. The x-axis represents the significance (p value) for each term, while the y-axis represents the ontology categories. ( C ) Real-time quantitative PCR was used to assess the expression of Runx2 in pre-hMSCs and ctrl-hMSCs. Runx2 mRNA expression was normalized to the control gene Gapdh and fold induction was then calculated in reference to ctrl-hMSCs. Results are expressed as mean ± SEM (n = 4).

    Techniques Used: Confocal Microscopy, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Significance Assay, Real-time Polymerase Chain Reaction, Expressing

    36) Product Images from "A subset of IL-17+ mesenchymal stem cells possesses anti-Candida albicans effect"

    Article Title: A subset of IL-17+ mesenchymal stem cells possesses anti-Candida albicans effect

    Journal: Cell Research

    doi: 10.1038/cr.2012.179

    Identification of IL-17 + MSCs. (A) Western blot analysis showed that human (h) and mouse (m) MSCs expressed IL-17, CD146 and Sca1, but not CD3 and CD4. Th17 cells were used as a positive control. (B) ELISA assay showed that hMSCs and mMSCs produced IL-17.
    Figure Legend Snippet: Identification of IL-17 + MSCs. (A) Western blot analysis showed that human (h) and mouse (m) MSCs expressed IL-17, CD146 and Sca1, but not CD3 and CD4. Th17 cells were used as a positive control. (B) ELISA assay showed that hMSCs and mMSCs produced IL-17.

    Techniques Used: Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Produced

    37) Product Images from "Smurf2-mediated degradation of EZH2 enhances neuron differentiation and improves functional recovery after ischaemic stroke"

    Article Title: Smurf2-mediated degradation of EZH2 enhances neuron differentiation and improves functional recovery after ischaemic stroke

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201783

    PPARγ agonist (rosiglitazone) can augment recovery of injuries by hMSC injection after cerebral ischaemia. A. Schematic representation for the investigation of chronic cerebral ischaemia with four therapeutic protocols (Group 1–4, bottom). Three cell implantation sites are located from anterior to caudal portion in the sagittal view of rat brain (top), and comparative injection sites (starred) as well as infarction areas (black) are in the coronal view of rat brain. B. A body asymmetry trial was used to assess body swing before and after middle cerebral artery ligation. Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, two-way ANOVA). C–E. Locomotor activities (vertical activity, vertical movement time, and the number of vertical movements) of all experimental rats were examined, Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, Two-way ANOVA). F. Grip strength measurement of the grasping power of forelimbs before (pre Tx) and 28 days after each of the three treatments (post Tx). Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, two-way ANOVA). G. Representative three-dimensional images of bisbenzimide-labelled hMSCs (blue fluorescence) implantation in rat brain. The white arrows indicate the implanted MAP2 (red fluorescence)-positive hMSCs in the ischaemic brains (top). Scale bars: 50 µm. Quantitative analysis of the implanted MAP2-positive cells numbers in both the hMSCs + rosiglitazone-treated rats and mock hMSCs-treated rats (bottom). Data are expressed as mean ± SEM. Significant p values are indicated ( n = 6, two-way ANOVA).
    Figure Legend Snippet: PPARγ agonist (rosiglitazone) can augment recovery of injuries by hMSC injection after cerebral ischaemia. A. Schematic representation for the investigation of chronic cerebral ischaemia with four therapeutic protocols (Group 1–4, bottom). Three cell implantation sites are located from anterior to caudal portion in the sagittal view of rat brain (top), and comparative injection sites (starred) as well as infarction areas (black) are in the coronal view of rat brain. B. A body asymmetry trial was used to assess body swing before and after middle cerebral artery ligation. Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, two-way ANOVA). C–E. Locomotor activities (vertical activity, vertical movement time, and the number of vertical movements) of all experimental rats were examined, Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, Two-way ANOVA). F. Grip strength measurement of the grasping power of forelimbs before (pre Tx) and 28 days after each of the three treatments (post Tx). Data are expressed as mean ± SEM. Significant p values are indicated ( n = 10, two-way ANOVA). G. Representative three-dimensional images of bisbenzimide-labelled hMSCs (blue fluorescence) implantation in rat brain. The white arrows indicate the implanted MAP2 (red fluorescence)-positive hMSCs in the ischaemic brains (top). Scale bars: 50 µm. Quantitative analysis of the implanted MAP2-positive cells numbers in both the hMSCs + rosiglitazone-treated rats and mock hMSCs-treated rats (bottom). Data are expressed as mean ± SEM. Significant p values are indicated ( n = 6, two-way ANOVA).

    Techniques Used: Injection, Ligation, Activity Assay, Fluorescence

    PPARγ accelerates neuron differentiation of hMSCs. Source data is available for this figure in the Supporting Information. A. qPCR analysis of lentiviral-mediated shRNA interference targeting PPARγ was used to allow for the generation of hMSC cells stably expressing shPPARγ (shPPARγ #B and #D). shLuc against luciferase was used as a negative control (mock). Error bars represent the SEM from three independent experiments ( n = 3). B. RT-PCR analysis of NSE (neuron marker) mRNA levels with or without PPARγ shRNAs (shPPARγ #B and #D) in 3A6-hMSCs after neuron differentiation. β-actin was used as an internal control. shLuc against luciferase was used as a negative control (mock). C. Immunoblotting of NeuN (neuron marker) protein expression with or without PPARγ shRNAs (shPPARγ #B and #D) in 3A6-hMSCs after neuron differentiation. D. Flow cytometric analysis of MSC marker (CD44) and neuron marker (MAP2) in 3A6-hMSCs with or without PPARγ knockdown at Day 0 and Day 5 after differentiation. The plots show the percentage of cells in undifferentiated (Day 0) and neuronal differentiated (Day 5) hMSCs (bottom). shLuc against luciferase was used as a negative control (mock). Error bars represent the SEM from three independent experiments. Significant p values are indicated ( n = 3, Student's t -test). E. The effect of PPARγ knockdown during neuron differentiation of 3A6-hMSCs and human primary MSCs (immunoblot, top). The plots (bottom) represent the relative density of MSC marker (CD105), neuron marker (MAP2) and PPARγ determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). F. The effect of the PPARγ agonist rosiglitazone during neuron differentiation of 3A6-hMSCs (immunoblot, top). The plots (bottom) represent the relative density of MSC marker (CD105) and neuron marker (MAP2) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). G. Immunoblot analysis of the MSC (CD105) and neuron (MAP2) markers in 3A6-hMSCs during neuron differentiation with ectopically expressed PPARγ (top). The plots (bottom) represent the relative density of MSC marker (CD105), neuron marker (MAP2) and PPARγ determined by scanning densitometric tracings. β-actin was used as an internal control. Error bars represent the SEM from three independent experiments ( n = 3).
    Figure Legend Snippet: PPARγ accelerates neuron differentiation of hMSCs. Source data is available for this figure in the Supporting Information. A. qPCR analysis of lentiviral-mediated shRNA interference targeting PPARγ was used to allow for the generation of hMSC cells stably expressing shPPARγ (shPPARγ #B and #D). shLuc against luciferase was used as a negative control (mock). Error bars represent the SEM from three independent experiments ( n = 3). B. RT-PCR analysis of NSE (neuron marker) mRNA levels with or without PPARγ shRNAs (shPPARγ #B and #D) in 3A6-hMSCs after neuron differentiation. β-actin was used as an internal control. shLuc against luciferase was used as a negative control (mock). C. Immunoblotting of NeuN (neuron marker) protein expression with or without PPARγ shRNAs (shPPARγ #B and #D) in 3A6-hMSCs after neuron differentiation. D. Flow cytometric analysis of MSC marker (CD44) and neuron marker (MAP2) in 3A6-hMSCs with or without PPARγ knockdown at Day 0 and Day 5 after differentiation. The plots show the percentage of cells in undifferentiated (Day 0) and neuronal differentiated (Day 5) hMSCs (bottom). shLuc against luciferase was used as a negative control (mock). Error bars represent the SEM from three independent experiments. Significant p values are indicated ( n = 3, Student's t -test). E. The effect of PPARγ knockdown during neuron differentiation of 3A6-hMSCs and human primary MSCs (immunoblot, top). The plots (bottom) represent the relative density of MSC marker (CD105), neuron marker (MAP2) and PPARγ determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). F. The effect of the PPARγ agonist rosiglitazone during neuron differentiation of 3A6-hMSCs (immunoblot, top). The plots (bottom) represent the relative density of MSC marker (CD105) and neuron marker (MAP2) determined by scanning densitometric tracings. Error bars represent the SEM from three independent experiments ( n = 3). G. Immunoblot analysis of the MSC (CD105) and neuron (MAP2) markers in 3A6-hMSCs during neuron differentiation with ectopically expressed PPARγ (top). The plots (bottom) represent the relative density of MSC marker (CD105), neuron marker (MAP2) and PPARγ determined by scanning densitometric tracings. β-actin was used as an internal control. Error bars represent the SEM from three independent experiments ( n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, shRNA, Stable Transfection, Expressing, Luciferase, Negative Control, Reverse Transcription Polymerase Chain Reaction, Marker

    38) Product Images from "Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors"

    Article Title: Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.1356

    Shockwave or ATP treatment induces osteogenic differentiation of human mesenchymal stem cells (hMSCs). ( A ): hMSCs were subjected to shockwave (0.18 mJ/mm 2 ) and then cultured for 14 or 30 days until analysis of alkaline phosphatase activity (14 days), osteocalcin (OC) protein production (30 days), and formation of bone nodules assessed by Alizarin Red staining (30 days). ( B ): Cells were stimulated by addition of exogenous ATP at the indicated final assay concentrations. After 5 minutes, the cells were plated and cultured for 14 or 30 days until analysis of alkaline phosphatase activity (14 days), OC protein production (30 days), and formation of bone nodules (30 days). Data represent means ± SE; *, p
    Figure Legend Snippet: Shockwave or ATP treatment induces osteogenic differentiation of human mesenchymal stem cells (hMSCs). ( A ): hMSCs were subjected to shockwave (0.18 mJ/mm 2 ) and then cultured for 14 or 30 days until analysis of alkaline phosphatase activity (14 days), osteocalcin (OC) protein production (30 days), and formation of bone nodules assessed by Alizarin Red staining (30 days). ( B ): Cells were stimulated by addition of exogenous ATP at the indicated final assay concentrations. After 5 minutes, the cells were plated and cultured for 14 or 30 days until analysis of alkaline phosphatase activity (14 days), OC protein production (30 days), and formation of bone nodules (30 days). Data represent means ± SE; *, p

    Techniques Used: Cell Culture, Activity Assay, Staining

    Shockwaves induce P2X7 receptor expression. ( A ): Expression of P2X7 receptor in human mesenchymal stem cells (hMSCs) (P4) was assessed by immunofluorescence staining with P2X7 receptor antibodies followed by fluorescence-labeled secondary antibodies. Control slides were processed in the same fashion, except that P2X7 receptor antibodies were omitted (up images). They were examined under a magnification of 400. ( B, C ): P2X7 receptor mRNA levels of hMSCs after repeated passages (panel B) or after shockwave or ATP treatments (panel C) were determined with real-time reverse transcription polymerase chain reaction. Cells were exposed to shockwave stimulation (100 impulses at 0.18 mJ/mm 2 ) or treatment with exogenous ATP (1 μ M) and P2X7 receptor mRNA expression was determined after 1 hour and expressed relative to β-actin control values. ( D ): P2X7 receptor protein levels in hMSC membrane preparations and β-actin levels in hMSC cytoplasm preparations were estimated by immunoblotting with P2X7 or β-actin antibodies and gray values were compared and expressed by normalizing to unstimulated controls. Data represent mean ± SE. *, p
    Figure Legend Snippet: Shockwaves induce P2X7 receptor expression. ( A ): Expression of P2X7 receptor in human mesenchymal stem cells (hMSCs) (P4) was assessed by immunofluorescence staining with P2X7 receptor antibodies followed by fluorescence-labeled secondary antibodies. Control slides were processed in the same fashion, except that P2X7 receptor antibodies were omitted (up images). They were examined under a magnification of 400. ( B, C ): P2X7 receptor mRNA levels of hMSCs after repeated passages (panel B) or after shockwave or ATP treatments (panel C) were determined with real-time reverse transcription polymerase chain reaction. Cells were exposed to shockwave stimulation (100 impulses at 0.18 mJ/mm 2 ) or treatment with exogenous ATP (1 μ M) and P2X7 receptor mRNA expression was determined after 1 hour and expressed relative to β-actin control values. ( D ): P2X7 receptor protein levels in hMSC membrane preparations and β-actin levels in hMSC cytoplasm preparations were estimated by immunoblotting with P2X7 or β-actin antibodies and gray values were compared and expressed by normalizing to unstimulated controls. Data represent mean ± SE. *, p

    Techniques Used: Expressing, Immunofluorescence, Staining, Fluorescence, Labeling, Reverse Transcription Polymerase Chain Reaction

    Shockwave treatment activates p38 MAPK via P2X7 receptor stimulation. ( A, B ): Shockwaves and exogenous ATP dose-dependently induce p38 MAPK activation. (A): After shockwave treatment (0.18 mJ/mm 2 ) with indicated impulse numbers, human mesenchymal stem cells (hMSCs) (10 6 per ml, P4) were cultured for 30 minutes, and p38 MAPK activation was determined by immunoblotting with antibodies that recognize the active phosphorylated form of p38 MAPK (p-p38) and antibodies that recognize both active and inactive p38 MAPK (pan-p38 MAPK; p38). Gray intensities were analyzed and ratios between activated and pan-p38 MAPK are calculated, and data were normalized by control nontreated group and present in the corresponding graphs. (B): hMSCs (10 6 per ml) were treated with the indicated concentrations of exogenous ATP for 5 minutes to simulate sample handling as was used for shockwave treatment. Then the cells were plated and cultured in the presence or absence of ATP for 30 minutes and p38 MAPK activation was determined. ( C, D ): Purinergic signaling via P2X7 receptors contributes to p38 MAPK activation. hMSCs were subjected to shockwave treatment (0.18 mJ/mm 2 for 100 impulses; panel (C) or extracellular ATP (1 μ M ATP for 5 minutes; panel (D)) in the absence or presence of a nonspecific P2 receptor antagonist (PPADS), specific antagonists of P2X7 (KN-62), P2Y1 receptors (MRS-2179), or apyrase, and p38 MAPK activation was determined. In order to silence P2X7 expression, cells were pretreated with small interfering RNA (siRNA) targeting P2X7 receptors or with control siRNA for 3 days prior to shockwave or ATP treatment. Representative Western blots of six different experiments are shown, and data were averaged in the corresponding bar graphs ( n = 6, mean ± SD, *, p
    Figure Legend Snippet: Shockwave treatment activates p38 MAPK via P2X7 receptor stimulation. ( A, B ): Shockwaves and exogenous ATP dose-dependently induce p38 MAPK activation. (A): After shockwave treatment (0.18 mJ/mm 2 ) with indicated impulse numbers, human mesenchymal stem cells (hMSCs) (10 6 per ml, P4) were cultured for 30 minutes, and p38 MAPK activation was determined by immunoblotting with antibodies that recognize the active phosphorylated form of p38 MAPK (p-p38) and antibodies that recognize both active and inactive p38 MAPK (pan-p38 MAPK; p38). Gray intensities were analyzed and ratios between activated and pan-p38 MAPK are calculated, and data were normalized by control nontreated group and present in the corresponding graphs. (B): hMSCs (10 6 per ml) were treated with the indicated concentrations of exogenous ATP for 5 minutes to simulate sample handling as was used for shockwave treatment. Then the cells were plated and cultured in the presence or absence of ATP for 30 minutes and p38 MAPK activation was determined. ( C, D ): Purinergic signaling via P2X7 receptors contributes to p38 MAPK activation. hMSCs were subjected to shockwave treatment (0.18 mJ/mm 2 for 100 impulses; panel (C) or extracellular ATP (1 μ M ATP for 5 minutes; panel (D)) in the absence or presence of a nonspecific P2 receptor antagonist (PPADS), specific antagonists of P2X7 (KN-62), P2Y1 receptors (MRS-2179), or apyrase, and p38 MAPK activation was determined. In order to silence P2X7 expression, cells were pretreated with small interfering RNA (siRNA) targeting P2X7 receptors or with control siRNA for 3 days prior to shockwave or ATP treatment. Representative Western blots of six different experiments are shown, and data were averaged in the corresponding bar graphs ( n = 6, mean ± SD, *, p

    Techniques Used: Activation Assay, Cell Culture, Expressing, Small Interfering RNA, Western Blot

    Shockwave treatment causes rapid ATP release and reduces hMSC viability. ( A ): Adherent hMSCs in a primary culture (P0) on days 4 and 14 after plating of freshly isolated bone marrow cells. ( B ): After passaging hMSCs four times (P4), their phenotype was analyzed by flow cytometry to distinguish hematopoietic (CD34 + , CD45 + ) and mesenchymal (CD73 + , CD105 + ) lineages. ( C ) After four passages, hMSCs (5 × 10 6 per ml) were exposed to shockwaves using the indicated impulse numbers. Cell viability was monitored with trypan blue 5 minutes after shockwave treatment (data in mean ± SD, n = 6, *, p
    Figure Legend Snippet: Shockwave treatment causes rapid ATP release and reduces hMSC viability. ( A ): Adherent hMSCs in a primary culture (P0) on days 4 and 14 after plating of freshly isolated bone marrow cells. ( B ): After passaging hMSCs four times (P4), their phenotype was analyzed by flow cytometry to distinguish hematopoietic (CD34 + , CD45 + ) and mesenchymal (CD73 + , CD105 + ) lineages. ( C ) After four passages, hMSCs (5 × 10 6 per ml) were exposed to shockwaves using the indicated impulse numbers. Cell viability was monitored with trypan blue 5 minutes after shockwave treatment (data in mean ± SD, n = 6, *, p

    Techniques Used: Isolation, Passaging, Flow Cytometry, Cytometry

    39) Product Images from "TLR3-/4-Priming Differentially Promotes Ca2+ Signaling and Cytokine Expression and Ca2+-Dependently Augments Cytokine Release in hMSCs"

    Article Title: TLR3-/4-Priming Differentially Promotes Ca2+ Signaling and Cytokine Expression and Ca2+-Dependently Augments Cytokine Release in hMSCs

    Journal: Scientific Reports

    doi: 10.1038/srep23103

    Exposure to LPS or Poly(I:C) elevates the mRNA expression of TLR3 and cytokines in hMSCs. ( a , b ) Conventional and real-time RT-PCR assays and quantification results showing that TLR3 and TLR4 mRNA expression levels were increased in a concentration and time dependent manner after exposure to LPS or poly(I:C). LPS (10 ng/ml) elevates TLR3 mRNA expression. Poly(I:C) concentration-dependently increases TLR3 mRNA expression. 4 h incubation with both ligands preferably elevated TLR3 and TLR4 mRNA levels. β-Actin serves as an internal control. ( c ) Real-time RT-PCR analysis revealing the 4 h incubation with LPS- and poly(I:C)-induced up-regulation of mRNA expression of cytokines including IL4, IL6, IL8 and IP10 in hMSCs. LPS preferably boosts IL6, IL8 and IP10 mRNA expression, whereas poly(I:C) raised only the mRNA level of IL4. The significance level was set at *p
    Figure Legend Snippet: Exposure to LPS or Poly(I:C) elevates the mRNA expression of TLR3 and cytokines in hMSCs. ( a , b ) Conventional and real-time RT-PCR assays and quantification results showing that TLR3 and TLR4 mRNA expression levels were increased in a concentration and time dependent manner after exposure to LPS or poly(I:C). LPS (10 ng/ml) elevates TLR3 mRNA expression. Poly(I:C) concentration-dependently increases TLR3 mRNA expression. 4 h incubation with both ligands preferably elevated TLR3 and TLR4 mRNA levels. β-Actin serves as an internal control. ( c ) Real-time RT-PCR analysis revealing the 4 h incubation with LPS- and poly(I:C)-induced up-regulation of mRNA expression of cytokines including IL4, IL6, IL8 and IP10 in hMSCs. LPS preferably boosts IL6, IL8 and IP10 mRNA expression, whereas poly(I:C) raised only the mRNA level of IL4. The significance level was set at *p

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Incubation

    Incubation with Poly(I:C) rather than LPS augments Orai and STIM expression and SOCE in hMSCs. ( a ) Averaged [Ca 2+ ] i traces showing [Ca 2+ ] i transients induced by stimulation with CPA (first) and those evoked by addition of extracellular Ca 2+ (second) in control (n = 40 cells), LPS- (n = 79 cells) and poly(I:C)-treated cells (n = 30 cells) immersed in Ca 2+ -free extracellular solution. ( b ) Summarized graph illustrating the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios recorded in control, LPS- or poly(I:C)-treated groups. Experiments were performed sixteen times. ( c ) Summarized graph showing the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios following extracellular application of 4 mM Ca 2+ in control, LPS- or poly(I:C)-treated cells with intracellular Ca 2+ stores pre-emptied by CPA. Experiments were performed sixteen times. ( d ) Representative RT-PCR blots (upper panel) illustrating the mRNA expression levels of three Orai subtypes and two STIM subtypes in control cells. NC represents the negative control with distilled water. Real-time RT-PCR quantification (lower panel) showing different mRNA expression profiles of three Orai subtypes (Orai1, Orai2 and Orai3) and two STIM subtypes (Stim1 and Stim2) in the control (n = 3), LPS (n = 3) and poly(I:C) (n = 3) groups. ( e ) Confocal images illustrating the different intensities of Orai1 and Orai2 immunofluorescence in control cells (upper panel) and cells exposed to LPS (middle panel) or poly(I:C) (lower panel). ( f ) Representative western blot of Orai2 in control cells and cells exposed to LPS or poly(I:C) (left panel). Summarized graph showing the normalized level of Orai2 in the indicated conditions. β-actin was used as a loading control. Experiments were performed four times (right panel). ( g ) Summarized graphs showing basal [Ca 2+ ] i reflected by the averaged F340/F380 ratios registered before application of CPA in control cells and cells exposed to LPS or poly(I:C). Experiments were performed nineteen times. The significance level was set at *p
    Figure Legend Snippet: Incubation with Poly(I:C) rather than LPS augments Orai and STIM expression and SOCE in hMSCs. ( a ) Averaged [Ca 2+ ] i traces showing [Ca 2+ ] i transients induced by stimulation with CPA (first) and those evoked by addition of extracellular Ca 2+ (second) in control (n = 40 cells), LPS- (n = 79 cells) and poly(I:C)-treated cells (n = 30 cells) immersed in Ca 2+ -free extracellular solution. ( b ) Summarized graph illustrating the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios recorded in control, LPS- or poly(I:C)-treated groups. Experiments were performed sixteen times. ( c ) Summarized graph showing the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios following extracellular application of 4 mM Ca 2+ in control, LPS- or poly(I:C)-treated cells with intracellular Ca 2+ stores pre-emptied by CPA. Experiments were performed sixteen times. ( d ) Representative RT-PCR blots (upper panel) illustrating the mRNA expression levels of three Orai subtypes and two STIM subtypes in control cells. NC represents the negative control with distilled water. Real-time RT-PCR quantification (lower panel) showing different mRNA expression profiles of three Orai subtypes (Orai1, Orai2 and Orai3) and two STIM subtypes (Stim1 and Stim2) in the control (n = 3), LPS (n = 3) and poly(I:C) (n = 3) groups. ( e ) Confocal images illustrating the different intensities of Orai1 and Orai2 immunofluorescence in control cells (upper panel) and cells exposed to LPS (middle panel) or poly(I:C) (lower panel). ( f ) Representative western blot of Orai2 in control cells and cells exposed to LPS or poly(I:C) (left panel). Summarized graph showing the normalized level of Orai2 in the indicated conditions. β-actin was used as a loading control. Experiments were performed four times (right panel). ( g ) Summarized graphs showing basal [Ca 2+ ] i reflected by the averaged F340/F380 ratios registered before application of CPA in control cells and cells exposed to LPS or poly(I:C). Experiments were performed nineteen times. The significance level was set at *p

    Techniques Used: Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Quantitative RT-PCR, Immunofluorescence, Western Blot

    Treatment with LPS or Poly(I:C) increases IP 3 R expression and IP 3 R-mediated Ca 2+ mobilization without influencing Dihydropyridine-Sensitive Ca 2+ entry in hMSCs. ( a ) Representative [Ca 2+ ] i traces showing carbachol-evoked [Ca 2+ ] i transients registered in four individual cells bathed in extracellular solution without Ca 2+ (left panel). Averaged [Ca 2+ ] i traces depicting the mean [Ca 2+ ] i responses to carbachol challenge in control cells (CTL; n = 26 cells) and cells treated with LPS (n = 43 cells) or poly(I:C) (n = 61 cells) in the absence of extracellular Ca 2+ . ( b ) Upper graph illustrating the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios obtained from control (CTL; n = 9), LPS- (n = 9) and poly(I:C)-treated groups (n = 9). Lower graph showing the averaged percentages of carbachol-responsive cells subjected to control treatment (n = 19), exposure to LPS (n = 19) and incubation with poly(I:C) (n = 19). Herein, n denotes the number of experiments. ( c ) Representative RT-PCR blots showing the mRNA expression of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) and two RyR subtypes (RYR1 and RYR2) in control cells. GAPDH serves as an internal control. NC indicates negative control, i.e., distilled water. Real time RT-PCR quantification illustrating the different mRNA expression profiles of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) in the control, LPS and poly(I:C) groups. Experiments were performed three times. ( d ) Confocal images showing the different intensities of IP 3 R3 immunofluorescence in control cells (left panel) and cells exposed to LPS (middle panel) or poly(I:C) (right panel). ( e ) Representative western blot of IP 3 R3 in control cells and cells exposed to LPS or poly(I:C) (left panel). Summarized graph showing the normalized level of IP 3 R in indicated conditions (right panel). Pan-Cadherin was used as a loading control. Experiments were performed six times. The significance level was set at *p
    Figure Legend Snippet: Treatment with LPS or Poly(I:C) increases IP 3 R expression and IP 3 R-mediated Ca 2+ mobilization without influencing Dihydropyridine-Sensitive Ca 2+ entry in hMSCs. ( a ) Representative [Ca 2+ ] i traces showing carbachol-evoked [Ca 2+ ] i transients registered in four individual cells bathed in extracellular solution without Ca 2+ (left panel). Averaged [Ca 2+ ] i traces depicting the mean [Ca 2+ ] i responses to carbachol challenge in control cells (CTL; n = 26 cells) and cells treated with LPS (n = 43 cells) or poly(I:C) (n = 61 cells) in the absence of extracellular Ca 2+ . ( b ) Upper graph illustrating the mean net increases in [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios obtained from control (CTL; n = 9), LPS- (n = 9) and poly(I:C)-treated groups (n = 9). Lower graph showing the averaged percentages of carbachol-responsive cells subjected to control treatment (n = 19), exposure to LPS (n = 19) and incubation with poly(I:C) (n = 19). Herein, n denotes the number of experiments. ( c ) Representative RT-PCR blots showing the mRNA expression of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) and two RyR subtypes (RYR1 and RYR2) in control cells. GAPDH serves as an internal control. NC indicates negative control, i.e., distilled water. Real time RT-PCR quantification illustrating the different mRNA expression profiles of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) in the control, LPS and poly(I:C) groups. Experiments were performed three times. ( d ) Confocal images showing the different intensities of IP 3 R3 immunofluorescence in control cells (left panel) and cells exposed to LPS (middle panel) or poly(I:C) (right panel). ( e ) Representative western blot of IP 3 R3 in control cells and cells exposed to LPS or poly(I:C) (left panel). Summarized graph showing the normalized level of IP 3 R in indicated conditions (right panel). Pan-Cadherin was used as a loading control. Experiments were performed six times. The significance level was set at *p

    Techniques Used: Expressing, CTL Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Quantitative RT-PCR, Immunofluorescence, Western Blot

    Stimulation with LPS or Poly(I:C) Promotes Cytokine Release in a Ca 2+ Dependent Manner in hMSCs. ( a ) ELISA assay revealing more pronounced releases of IL6, IL8, IP10 and RANTES from cells exposed to LPS or poly(I:C) in comparison with control cells. Experiments were performed three times. ( b – d ) ELISA assay demonstrating the ablation of IL6, RANTES and IFN-alpha release by chelation of intracellular Ca 2+ with BAPTA/AM (5 μM) and siRNA from LPS- or poly(I:C)-treated cells. Experiments were performed three times. ( e ) Real-time RT-PCR quantification showing ITPR3, Orai2 and Stim1 mRNA expression profiles in control and poly(I:C) with and without BAPTA/AM. Experiments were performed three times. ( f ) ELISA assay demonstrating the ablation of IL6 release by ITPR3 knockdown (ITPR3-siRNA). Experiments were performed six times. The significance level was set at *p
    Figure Legend Snippet: Stimulation with LPS or Poly(I:C) Promotes Cytokine Release in a Ca 2+ Dependent Manner in hMSCs. ( a ) ELISA assay revealing more pronounced releases of IL6, IL8, IP10 and RANTES from cells exposed to LPS or poly(I:C) in comparison with control cells. Experiments were performed three times. ( b – d ) ELISA assay demonstrating the ablation of IL6, RANTES and IFN-alpha release by chelation of intracellular Ca 2+ with BAPTA/AM (5 μM) and siRNA from LPS- or poly(I:C)-treated cells. Experiments were performed three times. ( e ) Real-time RT-PCR quantification showing ITPR3, Orai2 and Stim1 mRNA expression profiles in control and poly(I:C) with and without BAPTA/AM. Experiments were performed three times. ( f ) ELISA assay demonstrating the ablation of IL6 release by ITPR3 knockdown (ITPR3-siRNA). Experiments were performed six times. The significance level was set at *p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

    Characterization of TLR4-primed hMSCs. ( a ) Flow cytometry analysis represented the immunophenotype of hMSC. hMSCs expressed CD44, CD29, CD90, CD105 and CD73. ( b ) RT-PCR confirmation using stem cell marker genes. RT-PCR analysis used that stem cell markers OCT4, SOX2, OPN, CXCR4, and COL10A1. GAPDH was used as an endogenous control. ( c ) hMSC morphology in normal conditions (left) with 100X magnification. Differentiation potential into adipocytes (middle) or osteoblasts (right) was shown with 400X magnification. Adipocytes or osteoblasts were stained with FABP4 or osteocalcin antibody (green), and nuclei were counterstained with DAPI (blue).
    Figure Legend Snippet: Characterization of TLR4-primed hMSCs. ( a ) Flow cytometry analysis represented the immunophenotype of hMSC. hMSCs expressed CD44, CD29, CD90, CD105 and CD73. ( b ) RT-PCR confirmation using stem cell marker genes. RT-PCR analysis used that stem cell markers OCT4, SOX2, OPN, CXCR4, and COL10A1. GAPDH was used as an endogenous control. ( c ) hMSC morphology in normal conditions (left) with 100X magnification. Differentiation potential into adipocytes (middle) or osteoblasts (right) was shown with 400X magnification. Adipocytes or osteoblasts were stained with FABP4 or osteocalcin antibody (green), and nuclei were counterstained with DAPI (blue).

    Techniques Used: Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Marker, Staining

    40) Product Images from "A Fluidic Culture Platform for Spatially Patterned Cell Growth, Differentiation, and Cocultures"

    Article Title: A Fluidic Culture Platform for Spatially Patterned Cell Growth, Differentiation, and Cocultures

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2018.0020

    Spatial control of coculture of hMSCs and HUVECs in culture chambers. Seeding order determine the spatial distribution of the cells: hMSCs are localized near the pillars if seeded first (A-i) , while both cell types are more uniformly distributed throughout the chamber if ECs are seeded first (A-ii) . (A) Example of fluorescent images of hMSCs and HUVECs near pillars inside flow channel after 1 day of culture. Dashed white line indicates the edge of a pillar. (B) Cell density profile for when hMSCs are seeded first (i) and when HUVECs are seeded first (ii) . Black dotted line indicates mean of the distribution. Means of the distributions indicate statistical difference between dominant growth/attachments regions of hMSCs and HUVECs when the hMSCs were seeded first, however, show no statistical difference when the HUVECs were seeded first. Values are averages of four experiments and error bars are standard deviations (** p  ≤ 0.01). (C) Actin fibers configuration in hMSCs and HUVECs, and size-dependent cell attachment and localization. Red : F-actin, blue : nucleus, green : live cells. HUVECs localization is seen at corners with 60° angle, but not at 90° angle. EC, endothelial cell; HUVECs, human umbilical vein endothelial cells.
    Figure Legend Snippet: Spatial control of coculture of hMSCs and HUVECs in culture chambers. Seeding order determine the spatial distribution of the cells: hMSCs are localized near the pillars if seeded first (A-i) , while both cell types are more uniformly distributed throughout the chamber if ECs are seeded first (A-ii) . (A) Example of fluorescent images of hMSCs and HUVECs near pillars inside flow channel after 1 day of culture. Dashed white line indicates the edge of a pillar. (B) Cell density profile for when hMSCs are seeded first (i) and when HUVECs are seeded first (ii) . Black dotted line indicates mean of the distribution. Means of the distributions indicate statistical difference between dominant growth/attachments regions of hMSCs and HUVECs when the hMSCs were seeded first, however, show no statistical difference when the HUVECs were seeded first. Values are averages of four experiments and error bars are standard deviations (** p  ≤ 0.01). (C) Actin fibers configuration in hMSCs and HUVECs, and size-dependent cell attachment and localization. Red : F-actin, blue : nucleus, green : live cells. HUVECs localization is seen at corners with 60° angle, but not at 90° angle. EC, endothelial cell; HUVECs, human umbilical vein endothelial cells.

    Techniques Used: Flow Cytometry, Cell Attachment Assay

    Related Articles

    Activation Assay:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Expressing:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Marker:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    ALP Assay:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Flow Cytometry:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Cytometry:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Incubation:

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo
    Article Snippet: Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation. hMSC basic medium/control medium was composed of hMSC proliferative medium without basic FGF, hMSC osteogenic medium was composed of hMSC basic medium supplemented with 10−8 M dex (Sigma), and hMSC mineralization medium was composed of basic medium supplemented with 10−8 . .. To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods. ..

    Article Title: Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues
    Article Snippet: Primers were designed for VGLL4 , KLHL24 , RCBTB2 , SCARF2 , ARHGAP24 , ACAD10 , HEPH , and BDH2 . cDNA preparation and gene expression analysis were performed as described above in the RNA-interference section. .. For differentiation following siRNA transfection, hAMSCs were seeded at 3,000 cells per cm2 in maintenance medium in six-well plates and incubated under standard culture conditions for 24 h before being changed to osteogenic medium containing vehicle (DMSO) or 0.1 μg/mL CytoD (Sigma-Aldrich). ..

    MTT Assay:

    Article Title: Extremely low frequency electromagnetic fields promote mesenchymal stem cell migration by increasing intracellular Ca2+ and activating the FAK/Rho GTPases signaling pathways in vitro
    Article Snippet: Migratory cells were imaged and counted in high power microscope micro-photographs (field area: 0.98 mm2 ) taken under bright light (Olympus Tokyo, Japan) using Image Pro Plus 6.0 software (Rockville, MD). .. Cell proliferation The proliferation of human BM-MSCs was analyzed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma, St. Louis, MO) assay. .. Human BM-MSCs (2 × 103 ) in 200 μl Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS) were plated in 96-well culture plates.

    Transfection:

    Article Title: Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues
    Article Snippet: Primers were designed for VGLL4 , KLHL24 , RCBTB2 , SCARF2 , ARHGAP24 , ACAD10 , HEPH , and BDH2 . cDNA preparation and gene expression analysis were performed as described above in the RNA-interference section. .. For differentiation following siRNA transfection, hAMSCs were seeded at 3,000 cells per cm2 in maintenance medium in six-well plates and incubated under standard culture conditions for 24 h before being changed to osteogenic medium containing vehicle (DMSO) or 0.1 μg/mL CytoD (Sigma-Aldrich). ..

    Staining:

    Article Title: Transplantation of Heterospheroids of Islet Cells and Mesenchymal Stem Cells for Effective Angiogenesis and Antiapoptosis
    Article Snippet: .. hMSCs were stained with PKH26 (Sigma-Aldrich) before the spheroid formation. ..

    Cell Culture:

    Article Title: A Newly Identified Mechanism Involved in Regulation of Human Mesenchymal Stem Cells by Fibrous Substrate Stiffness
    Article Snippet: After additional wash, cellular substrates were incubated with an FITC-conjugated antibody detecting BrdU localized in cell nuclei (sc-32323, Santa Cruz Biotechnology, Santa Cruz, CA, USA). .. To investigate whether stiff substrates regulate the osteogenic potential of hMSCs by activating the AKT signaling pathway, hMSCs cultured on 75PLLA and control PLLA were treated with 0.1 μM AKT Inhibitor IV (EMD Millipore) for 6 h before analysis. .. To determine the role of MIF in the mechanism of our interest, hMSCs maintained in basal growth medium after 48 h were transfected with MIF siRNA (sc-37137; Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the Lipofectamine 2000 transfection reagent (Life Technologies) following the manufacturer’s protocol.

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    Millipore hmscs
    Fabrication and assembly of the OMA hydrogel beads and <t>hMSC-laden</t> 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 <t>hMSCs</t> 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.
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    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

    (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

    db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    doi: 10.1073/pnas.0711190105

    Figure Lengend Snippet: db-cAMP augments the in vivo bone-forming capacity of hMSCs. ( a ) hMSCs were cultured on BCP particles in basic medium (Con) or osteogenic medium (Dex) for 7 days and implanted s.c. in nude mice for 6 weeks. Histomorphometric analysis demonstrates that osteogenic medium does not affect in vivo bone formation. Note the amount of bone formed by an equal number of goat-derived MSCs (G-MSCs) in an independent experiment. ( b ) In vivo bone formation by hMSCs from three donors using the standard tissue engineering approach (see Materials and Methods ). ns, not significant. ( c ) Bone formation using the peroperative seeding approach. Note the consistent increase in bone formation upon db-cAMP treatment. The data from b and c were analyzed by using Student's t test compared with their respective controls. ( d ) Incidence of bone formation using the peroperative seeding approach by hMSCs from five donors. ( e ) In vivo bone formation by hMSCs cultured in a perfusion bioreactor system in proliferation medium (con) or proliferation medium supplemented with 1 mM db-cAMP (cAMP). The data were analyzed by using Student's t test. ( f ) A representative histological section showing newly formed bone (red), matrix-embedded osteocytes (white arrow), and lining osteoblasts (black arrow). ( g ) Bone marrow-like tissue was seen at multiple places in bone derived from db-cAMP-treated hMSCs (white arrow). *, P

    Article Snippet: To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods.

    Techniques: In Vivo, Cell Culture, Mouse Assay, Derivative Assay

    PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    doi: 10.1073/pnas.0711190105

    Figure Lengend Snippet: PKA activation induces in vitro osteogenesis of hMSCs. ( a ) Box plot showing the average percentage of ALP-positive cells from 14 donors in basic medium (Con), osteogenic medium (Dex), basic medium with 1 mM db-cAMP (cAMP), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). The data were analyzed by using two-way ANOVA followed by Dunnet's multiple-comparison test. Statistical significance is denoted compared with the control group. ( b ) hMSCs were grown in either mineralization medium (dex) or mineralization medium to which 1 mM db-cAMP was added during the first 3, 5, 10, 15, 25, or full 30 days after which calcium deposition was measured and expressed as micrograms of calcium per milliliter of sample. The data were analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test. ( c ) H89, a PKA inhibitor, reverses the db-cAMP-induced ALP expression. hMSCs were preincubated with H89 for 10–15 h and then cotreated with db-cAMP or cholera toxin (CTX) for 4 days. The data were analyzed by using one-way ANOVA followed by Tukey's multiple-comparison test. ( d ) Addition of db-cAMP to hMSCs for 6 h resulted in increased phosphorylation of transcription factor CREB, which could be inhibited by coincubation with H89. *, P

    Article Snippet: To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods.

    Techniques: Activation Assay, In Vitro, ALP Assay, Expressing

    Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    doi: 10.1073/pnas.0711190105

    Figure Lengend Snippet: Model for autocrine/paracrine induction of osteogenesis in hMSCs by PKA signaling. db-cAMP induces direct expression of BMP target genes such as ID-2 and ID-4 via CREB resulting in cell-autonomous stimulation of osteogenesis whereas expression of BMP-2, proosteogenic cytokines, and growth factors results in paracrine induction of bone formation.

    Article Snippet: To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods.

    Techniques: Expressing

    db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

    doi: 10.1073/pnas.0711190105

    Figure Lengend Snippet: db-cAMP-induced gene and protein expression. ( a ) hMSCs were treated with cycloheximide for 1 h and then coincubated with db-cAMP for 6 more hours. Expression of BMP target genes ID-1 and ID-2 was analyzed compared to cycloheximide-treated cells. The data were analyzed by using Student's t test. ( b ) hMSCs were grown in basic medium, basic medium supplemented with 1 mM db-cAMP (cAMP), osteogenic medium (Dex), or osteogenic medium supplemented with 1 mM db-cAMP (Dex+cAMP). Expression of ID-1 was analyzed by qPCR and is expressed as fold induction compared with cells grown in basic medium. The data were analyzed by using two-way ANOVA. Statistical differences are denoted compared with cells grown in basic medium. ( c and d ) db-cAMP induces secretion of proosteogenic cytokines and growth factors. hMSCs were treated with db-cAMP for 4 days, the supernatant was collected, and IGF-1 ( c ), IL-8, and IL-11 ( d ) expression in the medium was measured by ELISA. The data were analyzed by using Student's t test. **, P

    Article Snippet: To determine whether PKA activation elicits an osteogenic response in hMSCs, we exposed them to 1 mM db-cAMP (Sigma) with or without dex for 4 days and analyzed the expression of the osteogenic marker ALP by flow cytometry. hMSCs were seeded at 5,000 cells per square centimeter and allowed to attach for 10–15 h in basic medium, then cells were incubated with 10−8 M dex and 1 mM db-cAMP for the denoted time periods.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Histological cross-sections of scaffolds seeded with BCH and hMSCs stained by H E and Alcian blue at different days of culture in differentiation medium.

    Journal: PLoS ONE

    Article Title: Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering

    doi: 10.1371/journal.pone.0055451

    Figure Lengend Snippet: Histological cross-sections of scaffolds seeded with BCH and hMSCs stained by H E and Alcian blue at different days of culture in differentiation medium.

    Article Snippet: DNA quantification Scaffolds seeded with BCH and hMSCs in differentiation medium at 1, 14 and 35 days were washed with PBS and frozen at −80°C before proteinase K (Sigma Aldrich) digestion.

    Techniques: Staining

    DNA assay on the scaffolds seeded with BCH and hMSCs in differentiation medium. Significant differences between each cell type at different time points were found for p

    Journal: PLoS ONE

    Article Title: Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering

    doi: 10.1371/journal.pone.0055451

    Figure Lengend Snippet: DNA assay on the scaffolds seeded with BCH and hMSCs in differentiation medium. Significant differences between each cell type at different time points were found for p

    Article Snippet: DNA quantification Scaffolds seeded with BCH and hMSCs in differentiation medium at 1, 14 and 35 days were washed with PBS and frozen at −80°C before proteinase K (Sigma Aldrich) digestion.

    Techniques: