mscs  (Lonza)


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    Name:
    Trypsin EDTA for Mesenchymal Stem Cells
    Description:
    Trypsin EDTA for MSC 100 mL
    Catalog Number:
    CC-3232
    Price:
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    Category:
    Primary and Stem Cells
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    Structured Review

    Lonza mscs
    VSMC-induced MSC proliferation requires mitochondrial transfer from VSMCs to <t>MSCs.</t> Graph showing MFI represents the cell proliferation in CFSE-labeled MSCs and in <t>cocultures</t> with control VSMCs and with VSMCs having mitochondrial dysfunction. Cell proliferation
    Trypsin EDTA for MSC 100 mL
    https://www.bioz.com/result/mscs/product/Lonza
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mscs - by Bioz Stars, 2021-06
    96/100 stars

    Images

    1) Product Images from "Vascular Smooth Muscle Cells Initiate Proliferation of Mesenchymal Stem Cells by Mitochondrial Transfer via Tunneling Nanotubes"

    Article Title: Vascular Smooth Muscle Cells Initiate Proliferation of Mesenchymal Stem Cells by Mitochondrial Transfer via Tunneling Nanotubes

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2011.0691

    VSMC-induced MSC proliferation requires mitochondrial transfer from VSMCs to MSCs. Graph showing MFI represents the cell proliferation in CFSE-labeled MSCs and in cocultures with control VSMCs and with VSMCs having mitochondrial dysfunction. Cell proliferation
    Figure Legend Snippet: VSMC-induced MSC proliferation requires mitochondrial transfer from VSMCs to MSCs. Graph showing MFI represents the cell proliferation in CFSE-labeled MSCs and in cocultures with control VSMCs and with VSMCs having mitochondrial dysfunction. Cell proliferation

    Techniques Used: Labeling

    VSMC-induced MSC proliferation requires TNT formation. (a) Graph showing MFI represents the cell proliferation in CFSE-labeled MSCs and VSMC cocultures. Increase in MSC proliferation was completely abolished by TNT disruption with cytochalasin D (1 μM)
    Figure Legend Snippet: VSMC-induced MSC proliferation requires TNT formation. (a) Graph showing MFI represents the cell proliferation in CFSE-labeled MSCs and VSMC cocultures. Increase in MSC proliferation was completely abolished by TNT disruption with cytochalasin D (1 μM)

    Techniques Used: Labeling

    MSC coculture with VSMCs does not induce MSC differentiation. (a) Representative flow cytometric analysis shows no change of VSMC markers α-SMA and calponin in MSCs and VSMCs in mono- and cocultures; n =3. (b) Expression of VSMC markers in MSCs
    Figure Legend Snippet: MSC coculture with VSMCs does not induce MSC differentiation. (a) Representative flow cytometric analysis shows no change of VSMC markers α-SMA and calponin in MSCs and VSMCs in mono- and cocultures; n =3. (b) Expression of VSMC markers in MSCs

    Techniques Used: Flow Cytometry, Expressing

    MSCs and VSMCs form TNT-like structures for intercellular contacts. Flow cytometry of cocultured MSCs and VSMCs. MSCs were labeled with CellTracker ™ (CM-DiI), whereas VSMCs were unlabeled; cytochalasin D (1 μM) was used to disrupt
    Figure Legend Snippet: MSCs and VSMCs form TNT-like structures for intercellular contacts. Flow cytometry of cocultured MSCs and VSMCs. MSCs were labeled with CellTracker ™ (CM-DiI), whereas VSMCs were unlabeled; cytochalasin D (1 μM) was used to disrupt

    Techniques Used: Flow Cytometry, Cytometry, Labeling

    Intercellular exchange of mitochondria between MSCs and VSMCs. MSCs or VSMCs were labeled with either MitoTracker Red ( red ) or CFSE ( green ). MitoTracker Red-labeled cells were cocultured for 2 and 24 h with CFSE-labeled cells. Fluorescence confocal
    Figure Legend Snippet: Intercellular exchange of mitochondria between MSCs and VSMCs. MSCs or VSMCs were labeled with either MitoTracker Red ( red ) or CFSE ( green ). MitoTracker Red-labeled cells were cocultured for 2 and 24 h with CFSE-labeled cells. Fluorescence confocal

    Techniques Used: Labeling, Fluorescence

    2) Product Images from "The crosstalk between vascular MSCs and inflammatory mediators determines the pro-calcific remodelling of human atherosclerotic aneurysm"

    Article Title: The crosstalk between vascular MSCs and inflammatory mediators determines the pro-calcific remodelling of human atherosclerotic aneurysm

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0554-x

    Inflammation enhances the mineralization process in ha-MSCs. a After inflammatory stimulation, ha-MSCs were cultured with specific osteogenic and adipogenic induction media for 21 and 14 days, respectively. b Calcium mineralization process was significantly marked under inflammatory conditions, mainly after AAA-PBMC influence as showed by Alizarin Red staining. Quantification values are represented as mean ± standard deviation and compared with induced ha-MSCs. c MMP-9 detection on osteogenic differentiated ha-MSCs was performed by immunofluorescence, revealing an appreciable staining only after osteogenic induction; 20× magnification. d Oil Red O staining of lipid droplets in ha-MSCs was reduced after priming cells with inflammatory cytokines, as shown by e PPAR-γ mRNA. Results expressed as fold changes relative to induced ha-MSCs. * p
    Figure Legend Snippet: Inflammation enhances the mineralization process in ha-MSCs. a After inflammatory stimulation, ha-MSCs were cultured with specific osteogenic and adipogenic induction media for 21 and 14 days, respectively. b Calcium mineralization process was significantly marked under inflammatory conditions, mainly after AAA-PBMC influence as showed by Alizarin Red staining. Quantification values are represented as mean ± standard deviation and compared with induced ha-MSCs. c MMP-9 detection on osteogenic differentiated ha-MSCs was performed by immunofluorescence, revealing an appreciable staining only after osteogenic induction; 20× magnification. d Oil Red O staining of lipid droplets in ha-MSCs was reduced after priming cells with inflammatory cytokines, as shown by e PPAR-γ mRNA. Results expressed as fold changes relative to induced ha-MSCs. * p

    Techniques Used: Cell Culture, Staining, Standard Deviation, Immunofluorescence

    ha-MSCs exposed to inflammatory conditions assume a pathological phenotype. a PBMCs isolated from AAA patients showed a molecular signature reporting high levels of inflammatory cytokines (TNF-α and IL-1β) and reduced anti-inflammatory IL-10. Results are expressed as fold changes relative to healthy PBMCs. b According to the experimental design, ha-MSCs were exposed to inflammatory mediators (cytokines and PBMCs) for 24 hours, then investigated in terms of vascular remodelling and differentiation properties. ha-MSCs exposed to inflammation underwent increased transcription of ( c ) MMP-9 and ( d ) osteogenic lineage-specific markers (BMP-2, OPN, OCN), to the detriment of the adipogenic transcriptional factor PPAR-γ. Results are expressed as fold changes relative to unexposed ha-MSCs. * p
    Figure Legend Snippet: ha-MSCs exposed to inflammatory conditions assume a pathological phenotype. a PBMCs isolated from AAA patients showed a molecular signature reporting high levels of inflammatory cytokines (TNF-α and IL-1β) and reduced anti-inflammatory IL-10. Results are expressed as fold changes relative to healthy PBMCs. b According to the experimental design, ha-MSCs were exposed to inflammatory mediators (cytokines and PBMCs) for 24 hours, then investigated in terms of vascular remodelling and differentiation properties. ha-MSCs exposed to inflammation underwent increased transcription of ( c ) MMP-9 and ( d ) osteogenic lineage-specific markers (BMP-2, OPN, OCN), to the detriment of the adipogenic transcriptional factor PPAR-γ. Results are expressed as fold changes relative to unexposed ha-MSCs. * p

    Techniques Used: Isolation

    3) Product Images from "Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts"

    Article Title: Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts

    Journal: Bosnian Journal of Basic Medical Sciences

    doi: 10.17305/bjbms.2018.2633

    The expression level of osteocalcin or bone gamma-carboxyglutamic acid-containing protein ( BGLAP ) gene in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of osteocalcin gene expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest expression of osteocalcin gene was detected on day 14. The difference in gene expression was calculated using the 2-ΔΔCT method ( p
    Figure Legend Snippet: The expression level of osteocalcin or bone gamma-carboxyglutamic acid-containing protein ( BGLAP ) gene in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of osteocalcin gene expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest expression of osteocalcin gene was detected on day 14. The difference in gene expression was calculated using the 2-ΔΔCT method ( p

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    Overexpression of miR-210 in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of miR-210 expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210 and MSCs transfected with Scramble on days 7, 14 and 21. The highest expression level of miR-210 was detected on day 7. The difference in gene expression was calculated using the 2-ΔΔCT method ( p
    Figure Legend Snippet: Overexpression of miR-210 in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of miR-210 expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210 and MSCs transfected with Scramble on days 7, 14 and 21. The highest expression level of miR-210 was detected on day 7. The difference in gene expression was calculated using the 2-ΔΔCT method ( p

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation

    Mesenchymal stem cells (MSCs) were successfully transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210 by electroporation, (A) as observed under optical microscope. (B) Approximately 63% of transfected MSCs emitted green fluorescence under the inverted fluorescence microscope, 48 hours after the transfection. (C) Flow cytometric analysis also confirmed successful transfection of MSCs, i.e., infection rate of 63% was observed.
    Figure Legend Snippet: Mesenchymal stem cells (MSCs) were successfully transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210 by electroporation, (A) as observed under optical microscope. (B) Approximately 63% of transfected MSCs emitted green fluorescence under the inverted fluorescence microscope, 48 hours after the transfection. (C) Flow cytometric analysis also confirmed successful transfection of MSCs, i.e., infection rate of 63% was observed.

    Techniques Used: Transfection, Plasmid Preparation, Electroporation, Microscopy, Fluorescence, Flow Cytometry, Infection

    The expression level of runt-related transcription factor 2 (Runx2) in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of Runx2 expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest expression of Runx2 was detected on day 7. The difference in gene expression was calculated using the 2-ΔΔCT method ( p
    Figure Legend Snippet: The expression level of runt-related transcription factor 2 (Runx2) in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of Runx2 expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest expression of Runx2 was detected on day 7. The difference in gene expression was calculated using the 2-ΔΔCT method ( p

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The expression level of alkaline phosphatase (ALP, ALPL ) gene in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of ALP expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest ALP gene expression was detected on day 21. The difference in gene expression was calculated using the 2-ΔΔCT method ( p
    Figure Legend Snippet: The expression level of alkaline phosphatase (ALP, ALPL ) gene in mesenchymal stem cells (MSCs) on different days of transfection. Fold increase ± standard error of the mean (SEM) of ALP expression was compared between MSCs transfected with plenti-III-mir-green fluorescent protein (GFP) plasmid bearing pre-miR-210, MSCs transfected with Scramble and MSCs transfected with pmaxGFP vector on days 0, 7, 14 and 21. The highest ALP gene expression was detected on day 21. The difference in gene expression was calculated using the 2-ΔΔCT method ( p

    Techniques Used: Expressing, ALP Assay, Transfection, Plasmid Preparation

    4) Product Images from "Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells"

    Article Title: Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.142794

    Liver organoid differentiation in vitro and in vivo . (A) Cluster analysis of gene expression profiles of liver organoids ( n ) in the profile of LO-D2 that are shared with the PH and Li profiles (in A) and their expression in MSCs cultured alone, in HUVECs alone, in cell mixture at the time of plating of iPSCs+MSCs+HUVECs (liver organoid day 0, LO-D0), in hepatic-specified endoderm iPSCs (HE-iPS), in definitive endoderm iPSCs (DE-iPS) and in undifferentiated iPSCs (UD-iPS).
    Figure Legend Snippet: Liver organoid differentiation in vitro and in vivo . (A) Cluster analysis of gene expression profiles of liver organoids ( n ) in the profile of LO-D2 that are shared with the PH and Li profiles (in A) and their expression in MSCs cultured alone, in HUVECs alone, in cell mixture at the time of plating of iPSCs+MSCs+HUVECs (liver organoid day 0, LO-D0), in hepatic-specified endoderm iPSCs (HE-iPS), in definitive endoderm iPSCs (DE-iPS) and in undifferentiated iPSCs (UD-iPS).

    Techniques Used: In Vitro, In Vivo, Expressing, Cell Culture

    Protein analysis of the culture supernatants. At day 12 of culture, the supernatants of the lower chambers from HE-iPSCs/MSCs, HE-iPSCs/HUVECs, HE-iPSCs/MSCs+HUVECs, and HE-iPSCs were harvested and subjected to a SOMAscan assay in three biological replicates. The relative fluorescence unit (RFU) of each cell co-culture combination was normalized to the RFU of HE-iPSCs cultured alone. Of 1180 tested proteins, 228 showed an at least 3-fold increase. Cluster analysis using Pearson correlation clustered the proteins in two groups (dashed boxes): (1) cluster A with 44 proteins abundantly produced by MSCs and HUVECs, but downregulated in co-culture of MSCs+HUVECs; and (2) cluster B with 50 proteins abundantly produced by the co-culture of MSCs+HUVECs, but downregulated in MSCs and HUVECs cultured alone. The enrichment analysis by GSEA/MSigDB of each cluster showed that protein sets relate to late liver differentiation in cluster A and to hypoxic responses in cluster B. Each row represents a protein, with expression indicated by the color scale.
    Figure Legend Snippet: Protein analysis of the culture supernatants. At day 12 of culture, the supernatants of the lower chambers from HE-iPSCs/MSCs, HE-iPSCs/HUVECs, HE-iPSCs/MSCs+HUVECs, and HE-iPSCs were harvested and subjected to a SOMAscan assay in three biological replicates. The relative fluorescence unit (RFU) of each cell co-culture combination was normalized to the RFU of HE-iPSCs cultured alone. Of 1180 tested proteins, 228 showed an at least 3-fold increase. Cluster analysis using Pearson correlation clustered the proteins in two groups (dashed boxes): (1) cluster A with 44 proteins abundantly produced by MSCs and HUVECs, but downregulated in co-culture of MSCs+HUVECs; and (2) cluster B with 50 proteins abundantly produced by the co-culture of MSCs+HUVECs, but downregulated in MSCs and HUVECs cultured alone. The enrichment analysis by GSEA/MSigDB of each cluster showed that protein sets relate to late liver differentiation in cluster A and to hypoxic responses in cluster B. Each row represents a protein, with expression indicated by the color scale.

    Techniques Used: Fluorescence, Co-Culture Assay, Cell Culture, Produced, Expressing

    Hepatic differentiation of HE-iPSCs induced by paracrine signals of MSCs and/or HUVECs. (A) Two-chamber culture system using a Transwell micropore membrane insert to separate the upper and lower chambers. Hepatic-specified endoderm iPSCs (HE-iPSCs) were plated in the upper chamber and HUVECs and/or MSCs were plated in the lower chamber, while maintaining fluid communication. (B) Timecourse monitoring of albumin and A1AT production from HE-iPSCs in the culture supernatant, as quantified by ELISA ( n =8). Culture conditions include HE-iPSCs plated in the upper chamber and the other cell types in the lower chamber. * P
    Figure Legend Snippet: Hepatic differentiation of HE-iPSCs induced by paracrine signals of MSCs and/or HUVECs. (A) Two-chamber culture system using a Transwell micropore membrane insert to separate the upper and lower chambers. Hepatic-specified endoderm iPSCs (HE-iPSCs) were plated in the upper chamber and HUVECs and/or MSCs were plated in the lower chamber, while maintaining fluid communication. (B) Timecourse monitoring of albumin and A1AT production from HE-iPSCs in the culture supernatant, as quantified by ELISA ( n =8). Culture conditions include HE-iPSCs plated in the upper chamber and the other cell types in the lower chamber. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    5) Product Images from "The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair"

    Article Title: The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-019-1491-7

    Analysis of the neuroregenerative and neuroprotective effects of clinical MSCs cultured in MSCGM or NLRM. a Neuroregeneration assay in spinal cord neuron (seeded in transwell inserts) and MSCs (seeded in culture wells) cocultures incubated for 5 days. The density of GAP-43-positive immunoreactivity in the bottom side of the transwell following scrape injury was analyzed and is shown in the bar chart. Representative GAP-43-positive micrographs are shown (bar = 200 μm). b Neuroprotection test in spinal cord neuron cultures and MSC cocultures after H 2 O 2 (120 μM for one day) treatment. The apoptotic cells in neuron cultures after coculture with MSCs and H 2 O 2 treatment were processed for TUNEL staining, and representative micrographs are shown (bar = 200 μm). The bar chart shows the quantification of the TUNEL-positive cells. Data are presented as the mean ± SEM. ** p
    Figure Legend Snippet: Analysis of the neuroregenerative and neuroprotective effects of clinical MSCs cultured in MSCGM or NLRM. a Neuroregeneration assay in spinal cord neuron (seeded in transwell inserts) and MSCs (seeded in culture wells) cocultures incubated for 5 days. The density of GAP-43-positive immunoreactivity in the bottom side of the transwell following scrape injury was analyzed and is shown in the bar chart. Representative GAP-43-positive micrographs are shown (bar = 200 μm). b Neuroprotection test in spinal cord neuron cultures and MSC cocultures after H 2 O 2 (120 μM for one day) treatment. The apoptotic cells in neuron cultures after coculture with MSCs and H 2 O 2 treatment were processed for TUNEL staining, and representative micrographs are shown (bar = 200 μm). The bar chart shows the quantification of the TUNEL-positive cells. Data are presented as the mean ± SEM. ** p

    Techniques Used: Cell Culture, Incubation, TUNEL Assay, Staining

    Characterization of the CM derived from 2 kinds of cultivated MSCs, grouped by donor age and by cytokine array analysis. a Hierarchical clustering of the rows and columns was carried out according to the cytokine array intensity values (calculated by ImageJ software) for MSCGM-CM (M) and NRLM-CM (N) samples from the different patients (young, middle-aged, and older) using MORPHEUS one minus Pearson correlation (Versatile matrix visualization and analysis software; https://software.broadinstitute.org/morpheus ). b The statistical graph shows that the levels of 6 out of 120 proteins/cytokines were significantly different between the 2 kinds of cultivated MSC-CM. A human cytokine antibody array kit from RayBiotech was used to analyze the MSC-CM, revealing the presence of 120 cytokines and trophic factors. Data are presented as the mean ± SEM. * p
    Figure Legend Snippet: Characterization of the CM derived from 2 kinds of cultivated MSCs, grouped by donor age and by cytokine array analysis. a Hierarchical clustering of the rows and columns was carried out according to the cytokine array intensity values (calculated by ImageJ software) for MSCGM-CM (M) and NRLM-CM (N) samples from the different patients (young, middle-aged, and older) using MORPHEUS one minus Pearson correlation (Versatile matrix visualization and analysis software; https://software.broadinstitute.org/morpheus ). b The statistical graph shows that the levels of 6 out of 120 proteins/cytokines were significantly different between the 2 kinds of cultivated MSC-CM. A human cytokine antibody array kit from RayBiotech was used to analyze the MSC-CM, revealing the presence of 120 cytokines and trophic factors. Data are presented as the mean ± SEM. * p

    Techniques Used: Derivative Assay, Software, Ab Array

    Characterization of clinical bone marrow MSCs expanded in MSCGM or NRLM. a Representative bright-field micrographs of clinical MSCs cultivated in MSCGM or NRLM. b Flow cytometry analysis of cell surface marker expression, including CD29, CD34, CD44, CD45, CD90, CD73, CD105, and CD166, in MSCGM- or NRLM-cultured MSCs. c Representative micrographs showing adipocyte differentiation potential (oil red-positive staining) from two kinds of cultivated MSCs. d Analysis of the CD90+ population in clinical MSCs cultivated in MSCGM or NRLM by flow cytometry ( N = 31, including commercial MSC samples shown as yellow spots). Data are presented as the mean ± SEM. *** p
    Figure Legend Snippet: Characterization of clinical bone marrow MSCs expanded in MSCGM or NRLM. a Representative bright-field micrographs of clinical MSCs cultivated in MSCGM or NRLM. b Flow cytometry analysis of cell surface marker expression, including CD29, CD34, CD44, CD45, CD90, CD73, CD105, and CD166, in MSCGM- or NRLM-cultured MSCs. c Representative micrographs showing adipocyte differentiation potential (oil red-positive staining) from two kinds of cultivated MSCs. d Analysis of the CD90+ population in clinical MSCs cultivated in MSCGM or NRLM by flow cytometry ( N = 31, including commercial MSC samples shown as yellow spots). Data are presented as the mean ± SEM. *** p

    Techniques Used: Flow Cytometry, Cytometry, Marker, Expressing, Cell Culture, Staining

    6) Product Images from "Human mesenchymal stromal cells as cellular drug-delivery vectors for glioblastoma therapy: a good deal?"

    Article Title: Human mesenchymal stromal cells as cellular drug-delivery vectors for glioblastoma therapy: a good deal?

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0605-2

    SFN content in primed MSCs and control of their viability. a Determination of the SFN content of primed MSCs. MSCs were incubated for 1 h at 37 °C with SFN (20 μM or 100 μM). After washing with HBSS, the SFN content of the cell pellet was determined by HPLC. b Determination of the viability of SFN-primed MSCs. MSCs were incubated for 1 h at 37 °C with or without SFN (20 μM or 100 μM). After washing with HBSS, cells were seeded in a 96-well plate. Cell survival was estimated after 24 h, with the CyQUANT® cell proliferation assay kit. The results obtained for unprimed MSCs were considered to correspond to 100% survival. Data are expressed as the mean of three independent experiments ± SEM. Asterisks (*) indicate significant differences from unprimed MSCs ( p
    Figure Legend Snippet: SFN content in primed MSCs and control of their viability. a Determination of the SFN content of primed MSCs. MSCs were incubated for 1 h at 37 °C with SFN (20 μM or 100 μM). After washing with HBSS, the SFN content of the cell pellet was determined by HPLC. b Determination of the viability of SFN-primed MSCs. MSCs were incubated for 1 h at 37 °C with or without SFN (20 μM or 100 μM). After washing with HBSS, cells were seeded in a 96-well plate. Cell survival was estimated after 24 h, with the CyQUANT® cell proliferation assay kit. The results obtained for unprimed MSCs were considered to correspond to 100% survival. Data are expressed as the mean of three independent experiments ± SEM. Asterisks (*) indicate significant differences from unprimed MSCs ( p

    Techniques Used: Incubation, High Performance Liquid Chromatography, CyQUANT Assay, Proliferation Assay

    SFN release from SFN-primed MSCs and in vitro toxicity of SFN-primed MSCs to U87MG cells and HUVECs. a Profile of in vitro SFN release by SFN-primed MSCs. b and c Viability of U87MG cells and HUVECs following exposure to SFN or SFN-primed MSCs. Two doses of MSCs were tested: 6 × 10 5 and 2 × 10 5 cells, corresponding to the release of about 3.2 μg and 1.1 μg SFN, respectively. The results obtained for U87MG cells and HUVECs cultured with culture medium alone were considered to correspond to 100% survival. Data are expressed as the mean of four wells ± SEM ( n = 2) (* p
    Figure Legend Snippet: SFN release from SFN-primed MSCs and in vitro toxicity of SFN-primed MSCs to U87MG cells and HUVECs. a Profile of in vitro SFN release by SFN-primed MSCs. b and c Viability of U87MG cells and HUVECs following exposure to SFN or SFN-primed MSCs. Two doses of MSCs were tested: 6 × 10 5 and 2 × 10 5 cells, corresponding to the release of about 3.2 μg and 1.1 μg SFN, respectively. The results obtained for U87MG cells and HUVECs cultured with culture medium alone were considered to correspond to 100% survival. Data are expressed as the mean of four wells ± SEM ( n = 2) (* p

    Techniques Used: In Vitro, Cell Culture

    Effect of SFN on cell survival. U87MG cells, HUVECs and MSCs were seeded in their standard growth media and were treated with various concentrations of SFN. Cell survival was estimated with the CyQUANT® cell proliferation assay kit. The results obtained for U87MG cells, HUVECs or MSCs cultured with culture medium alone were considered to correspond to 100% survival. Data are expressed as the mean of four wells ± SEM ( n = 3)
    Figure Legend Snippet: Effect of SFN on cell survival. U87MG cells, HUVECs and MSCs were seeded in their standard growth media and were treated with various concentrations of SFN. Cell survival was estimated with the CyQUANT® cell proliferation assay kit. The results obtained for U87MG cells, HUVECs or MSCs cultured with culture medium alone were considered to correspond to 100% survival. Data are expressed as the mean of four wells ± SEM ( n = 3)

    Techniques Used: CyQUANT Assay, Proliferation Assay, Cell Culture

    Effect of intranasal administrations of SFN, or of unprimed or SFN-primed MSCs on U87MG growth and angiogenesis. a Representation of the treatment protocol applied to U87MG-bearing mice. b Tumor volume distribution in each group, calculated by MRI on day 17. c Immunofluorescence staining for Ki67 and CD31 in the tumor on day 17 (scale bar = 100 μm). d and e Quantitative results for Ki67 and CD31 immunofluorescence. Results are expressed as the mean number of Ki67 + cells ( d ) or CD31 + vessels ( e ) per mm 2 ± SEM. (ǂ p
    Figure Legend Snippet: Effect of intranasal administrations of SFN, or of unprimed or SFN-primed MSCs on U87MG growth and angiogenesis. a Representation of the treatment protocol applied to U87MG-bearing mice. b Tumor volume distribution in each group, calculated by MRI on day 17. c Immunofluorescence staining for Ki67 and CD31 in the tumor on day 17 (scale bar = 100 μm). d and e Quantitative results for Ki67 and CD31 immunofluorescence. Results are expressed as the mean number of Ki67 + cells ( d ) or CD31 + vessels ( e ) per mm 2 ± SEM. (ǂ p

    Techniques Used: Mouse Assay, Magnetic Resonance Imaging, Immunofluorescence, Staining

    7) Product Images from "CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells"

    Article Title: CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells

    Journal: BioResearch Open Access

    doi: 10.1089/biores.2014.0029

    The effect of growth factor addition to unsorted MSC cocultures upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with MSCs culture conditions with supplemental growth factors (+GF) or without (–GF). SMCs cocultures (GF) and EOC monocultures (+GF) were used as controls. (B) EOC network morphology was observed over 14 days of coculture and quantified. Scale bar equals 200 μm. # Indicates p
    Figure Legend Snippet: The effect of growth factor addition to unsorted MSC cocultures upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with MSCs culture conditions with supplemental growth factors (+GF) or without (–GF). SMCs cocultures (GF) and EOC monocultures (+GF) were used as controls. (B) EOC network morphology was observed over 14 days of coculture and quantified. Scale bar equals 200 μm. # Indicates p

    Techniques Used: In Vitro, Transduction, Expressing

    Evaluation of the effect of CD45-expressing cells within MSC populations, upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with either CD45- MSCs, unsorted MSCs, or SMCs for observation over 14 days of coculture. Scale bar equals 200 μm. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis for total segment length, mean segment length, and number of branch points. * p
    Figure Legend Snippet: Evaluation of the effect of CD45-expressing cells within MSC populations, upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with either CD45- MSCs, unsorted MSCs, or SMCs for observation over 14 days of coculture. Scale bar equals 200 μm. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis for total segment length, mean segment length, and number of branch points. * p

    Techniques Used: Expressing, In Vitro, Transduction

    Comparison of mesenchymal stem cell (MSC) and smooth muscle cell (SMC) coculture upon endothelial outgrowth cell (EOC) network formation in vitro . Images are representative of three independent experiments. Scale bars equal 100 μm. (A) EOCs were transduced with tomato-expressing lentivirus and combined with either MSCs or SMCs for observation over 18 days of coculture. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis of the number of branch points (B) and mean segment length (C) . (D, E) EOCs in MSC cocultures underwent cell loss over 10 days of culture in contrast to SMCs as evidenced by the percentage of platelet endothelial cell adhesion molecule (PECAM)–positive cells, normalized to total cell number. Image area analyzed is 0.57 mm 2 with n =3 images analyzed per condition. *Indicates p
    Figure Legend Snippet: Comparison of mesenchymal stem cell (MSC) and smooth muscle cell (SMC) coculture upon endothelial outgrowth cell (EOC) network formation in vitro . Images are representative of three independent experiments. Scale bars equal 100 μm. (A) EOCs were transduced with tomato-expressing lentivirus and combined with either MSCs or SMCs for observation over 18 days of coculture. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis of the number of branch points (B) and mean segment length (C) . (D, E) EOCs in MSC cocultures underwent cell loss over 10 days of culture in contrast to SMCs as evidenced by the percentage of platelet endothelial cell adhesion molecule (PECAM)–positive cells, normalized to total cell number. Image area analyzed is 0.57 mm 2 with n =3 images analyzed per condition. *Indicates p

    Techniques Used: In Vitro, Transduction, Expressing

    (A) Measured supernatant levels for thrombospondin-1 (TSP-1), interferon-gamma (IFN-γ), and interferon-alpha (IFN-α) in cocultures of EOCs with MSCs or SMCs during the first 18 days of culture. (B) Analysis of phagocytosis response within EOC and MSC cocultures during the first 10 days of culture; n =4 wells analyzed per condition. (C) Representative phagocytosis assay images depicting intracellular, fluorescein-labeled Escherichia coli within MSC cocultures during the first 10 days of coculture. Positive controls consisted of macrophages and negative controls consisted of experimental wells containing only Dulbecco's modified Eagle's medium. Scale bar equals 200 μm.
    Figure Legend Snippet: (A) Measured supernatant levels for thrombospondin-1 (TSP-1), interferon-gamma (IFN-γ), and interferon-alpha (IFN-α) in cocultures of EOCs with MSCs or SMCs during the first 18 days of culture. (B) Analysis of phagocytosis response within EOC and MSC cocultures during the first 10 days of culture; n =4 wells analyzed per condition. (C) Representative phagocytosis assay images depicting intracellular, fluorescein-labeled Escherichia coli within MSC cocultures during the first 10 days of coculture. Positive controls consisted of macrophages and negative controls consisted of experimental wells containing only Dulbecco's modified Eagle's medium. Scale bar equals 200 μm.

    Techniques Used: Phagocytosis Assay, Labeling, Modification

    8) Product Images from "Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine"

    Article Title: Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0936-8

    Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. ** p
    Figure Legend Snippet: Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. ** p

    Techniques Used: Activity Assay, Blocking Assay, ALP Assay

    Effect of conditioned medium from different cell types on platelet activation. Platelets incubated with either cell culture or conditioned medium (CM) and then stimulated by TRAP-6. Platelet activation measured by flow cytometry, assessing expression of CD62P, CD63 and PAC-1 binding. a Bone marrow (BM)-MSCs. b Lipoaspirate (LA)-MSCs. c Cord blood (CB)-MSCs. d Human umbilical vein endothelial cells (HUVECs). e HeLa tumor cells. Scale indicates relative activation marker expression of CM compared to culture medium. n = 3 biological replicates; HeLa cells n = 1, note different y axis for HeLa cells. * p
    Figure Legend Snippet: Effect of conditioned medium from different cell types on platelet activation. Platelets incubated with either cell culture or conditioned medium (CM) and then stimulated by TRAP-6. Platelet activation measured by flow cytometry, assessing expression of CD62P, CD63 and PAC-1 binding. a Bone marrow (BM)-MSCs. b Lipoaspirate (LA)-MSCs. c Cord blood (CB)-MSCs. d Human umbilical vein endothelial cells (HUVECs). e HeLa tumor cells. Scale indicates relative activation marker expression of CM compared to culture medium. n = 3 biological replicates; HeLa cells n = 1, note different y axis for HeLa cells. * p

    Techniques Used: Activation Assay, Incubation, Cell Culture, Flow Cytometry, Cytometry, Expressing, Binding Assay, Marker

    Expression and ectonucleotidase activity of CD39, CD73 and A2AR. a Cells stained with respective antibodies and MFI values determined by flow cytometry. BM-MSCs n = 3, LA-MSCs n = 7, CB-MSCs n = 5, HUVECs, n = 6 each biological replicates; HeLa n = 3 technical replicates; Plt unst, Plt stim each n = 4 biological replicates. * p
    Figure Legend Snippet: Expression and ectonucleotidase activity of CD39, CD73 and A2AR. a Cells stained with respective antibodies and MFI values determined by flow cytometry. BM-MSCs n = 3, LA-MSCs n = 7, CB-MSCs n = 5, HUVECs, n = 6 each biological replicates; HeLa n = 3 technical replicates; Plt unst, Plt stim each n = 4 biological replicates. * p

    Techniques Used: Expressing, Activity Assay, Staining, Flow Cytometry, Cytometry

    9) Product Images from "Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures"

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2019.00338

    Expression of the immunological molecule IDO. (A) RT-PCR analysis of IDO gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Relative gene expression levels of IDO normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) Left: Western blot analysis of IDO protein expression stimulated with IFN-γ/TNF-α (+) or unstimulated (–) after 48 h. An internal standard of IDO ranging from 25 to 200 ng was included. One representative blot of three independent experiments is shown. Right: Comparison of IDO protein expression in ng per 1 μg total protein of three IFN-γ/TNF-α stimulated batches.
    Figure Legend Snippet: Expression of the immunological molecule IDO. (A) RT-PCR analysis of IDO gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Relative gene expression levels of IDO normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) Left: Western blot analysis of IDO protein expression stimulated with IFN-γ/TNF-α (+) or unstimulated (–) after 48 h. An internal standard of IDO ranging from 25 to 200 ng was included. One representative blot of three independent experiments is shown. Right: Comparison of IDO protein expression in ng per 1 μg total protein of three IFN-γ/TNF-α stimulated batches.

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

    Examination of elasticity. (A) Left: Topographic images of membrane structures from MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL from passage 1 and 3 determined by atomic force microscopy. Middle: Elasticity mapping. Right: Surface elasticity measurements given by the Young's moduli shown in a histogram. (B) Comparison of 1,024 single surface elasticity measurements from individual MSCs shown in a box plot diagram ( n = 6).
    Figure Legend Snippet: Examination of elasticity. (A) Left: Topographic images of membrane structures from MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL from passage 1 and 3 determined by atomic force microscopy. Middle: Elasticity mapping. Right: Surface elasticity measurements given by the Young's moduli shown in a histogram. (B) Comparison of 1,024 single surface elasticity measurements from individual MSCs shown in a box plot diagram ( n = 6).

    Techniques Used: Cell Culture, Microscopy

    Mitochondrial pattern analysis and super-resolution imaging. (A) Images of Mitochondrial Network Analysis (MiNA) from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with MitoTracker™ (B) Top: Numbers of punctate mitochondrial organelles per cell compared in different media and from passage 1 and 3, Wilcoxon and Mann Whitney test. Middle: Numbers of mitochondrial rods per cell, Mann Whitney test. Bottom: Numbers of mitochondrial networks per cell, Wilcoxon test. MiNA image interpretations are shown next to the diagrams. (C) Left: Median branch length of the mitochondrial networks in μm per cell, paired and unpaired t -test. Right: Mitochondrial footprint in μm 2 per cell. The same 30 cells per media or passage were compared in (B,C) . (D) Reconstructed fluorescence microscopy images (dSTORM) of mitochondria from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL. (E) Numbers of punctate mitochondrial organelles as well as rods and networks per cell from passage 3 in different media. Data were calculated with Mitochondrial Network Analysis (MiNA), image interpretations are shown next to the diagrams. * p
    Figure Legend Snippet: Mitochondrial pattern analysis and super-resolution imaging. (A) Images of Mitochondrial Network Analysis (MiNA) from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with MitoTracker™ (B) Top: Numbers of punctate mitochondrial organelles per cell compared in different media and from passage 1 and 3, Wilcoxon and Mann Whitney test. Middle: Numbers of mitochondrial rods per cell, Mann Whitney test. Bottom: Numbers of mitochondrial networks per cell, Wilcoxon test. MiNA image interpretations are shown next to the diagrams. (C) Left: Median branch length of the mitochondrial networks in μm per cell, paired and unpaired t -test. Right: Mitochondrial footprint in μm 2 per cell. The same 30 cells per media or passage were compared in (B,C) . (D) Reconstructed fluorescence microscopy images (dSTORM) of mitochondria from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL. (E) Numbers of punctate mitochondrial organelles as well as rods and networks per cell from passage 3 in different media. Data were calculated with Mitochondrial Network Analysis (MiNA), image interpretations are shown next to the diagrams. * p

    Techniques Used: Imaging, Cell Culture, Staining, MANN-WHITNEY, Fluorescence, Microscopy

    Expression of the immunological molecule GARP. (A) Relative gene expression levels of GARP after 24 and 48 h cultivation with MSCGM™ or MSCBM™ with 8% HPL and a control and a control at the beginning of cultivation time. Left: Normalized to the housekeeping gene HPRT (2D n = 5; 3D n = 3). Right: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) RT-PCR analysis of GARP gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (C) Left: GARP measurements of living CD90 + MSCs in 2D vs. 3D cultured in different media determined by flow cytometry, 6,000 cells were compared. Right: Dot plot of the mean fluorescence intensity of three batches in 2D and four batches in 3D, 6,000 MSCs were investigated per batch. (D) Box plot of the mean fluorescence intensity of GARP protein expression of five batches unstimulated or stimulated with IFN-γ/TNF-α for 24 and 48 h.
    Figure Legend Snippet: Expression of the immunological molecule GARP. (A) Relative gene expression levels of GARP after 24 and 48 h cultivation with MSCGM™ or MSCBM™ with 8% HPL and a control and a control at the beginning of cultivation time. Left: Normalized to the housekeeping gene HPRT (2D n = 5; 3D n = 3). Right: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) RT-PCR analysis of GARP gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (C) Left: GARP measurements of living CD90 + MSCs in 2D vs. 3D cultured in different media determined by flow cytometry, 6,000 cells were compared. Right: Dot plot of the mean fluorescence intensity of three batches in 2D and four batches in 3D, 6,000 MSCs were investigated per batch. (D) Box plot of the mean fluorescence intensity of GARP protein expression of five batches unstimulated or stimulated with IFN-γ/TNF-α for 24 and 48 h.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    Effect of HPL on 2D and 3D cultures. (A) Actin measurements with phalloidin of living CD90 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of four different batches for 3D MSCs and five batches for 2D MSCs of 4,000 cells per batch, one representative flow cytometry histogram is shown. (B) Mitochondria measurements with MitoTracker™ of living CD73 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of five different batches, 2,000 MSCs were investigated per batch, one representative flow cytometry histogram is shown. (C) Images of passage 1 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with the specific focal adhesion proteins vinculin and paxillin in green as well as stress fibers (f-actin) in red and nuclei in blue. Mitochondria were stained with MitoTracker™ in red, stress fibers in green and nuclei in blue. Type-3 intermediate filaments are given by staining vimentin in green, stress fibers in red and nuclei in blue. (D) Images of MSC spheroids, the staining corresponds to those described in (A) . The entire MSC spheroid is a maximal intensity projection of a stitched z-stack 3-channel overlay, magnification was 63x.
    Figure Legend Snippet: Effect of HPL on 2D and 3D cultures. (A) Actin measurements with phalloidin of living CD90 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of four different batches for 3D MSCs and five batches for 2D MSCs of 4,000 cells per batch, one representative flow cytometry histogram is shown. (B) Mitochondria measurements with MitoTracker™ of living CD73 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of five different batches, 2,000 MSCs were investigated per batch, one representative flow cytometry histogram is shown. (C) Images of passage 1 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with the specific focal adhesion proteins vinculin and paxillin in green as well as stress fibers (f-actin) in red and nuclei in blue. Mitochondria were stained with MitoTracker™ in red, stress fibers in green and nuclei in blue. Type-3 intermediate filaments are given by staining vimentin in green, stress fibers in red and nuclei in blue. (D) Images of MSC spheroids, the staining corresponds to those described in (A) . The entire MSC spheroid is a maximal intensity projection of a stitched z-stack 3-channel overlay, magnification was 63x.

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

    10) Product Images from "Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock"

    Article Title: Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025171

    MSCs in the lung vasculature. Figures 6A-C show, IV MSCs localize to the lung vasculature and preserve integrity of AJs and tight junctions (TJs). Figure 6A , MSCs (see white arrow) were detected around blood vessels in the lungs (BVL = blood vessel lumen) using a human mitochondrial antibody (green) showing staining in lung tissue from MSC treated rats. (red = VE-Cadherin and DAPI staining nuclei -blue). Figure 6B: Staining of blood vessels in lungs (BVL = blood vessel lumen) for VE-Cadherin (red) and β-catenin (green) shows that in the LR group, HS and resuscitation compromise the continuity of the AJs (see white arrow). This continuity is preserved in MSC treated animals. Figure 6C: Similar findings are found for TJs staining of Occludin-1 (green) and Claudin-1 (red). MSCs preserve compromise of TJs in the lungs (see white arrows). Morphological changes (thickening) in AJs and TJs are noted in MSC treated lung vessels (long white arrows). Figures 6D–H show MSCs preserve PDGFRβ positive pericytes on lung microvasculature after HS. Sectioned lung tissue was stained for PDGFRβ (green) to identify pericytes or smooth muscle (SM) cells and vWF (red) to identify blood vessels. Figures 6E and 6F shows that HS and LR groups show diminished or compromised PDGFRβ staining (small white arrows), indicating decreased pericyte/SM cell coverage on microvasculature. This is increased above Sham ( Figure 6D ) in MSC treated animals ( Figure 6G -see large white arrow). Figure 6H shows that MSCs (red) contribute to some, but not all, of the increased PDGFRβ (green) staining found in treated lungs.
    Figure Legend Snippet: MSCs in the lung vasculature. Figures 6A-C show, IV MSCs localize to the lung vasculature and preserve integrity of AJs and tight junctions (TJs). Figure 6A , MSCs (see white arrow) were detected around blood vessels in the lungs (BVL = blood vessel lumen) using a human mitochondrial antibody (green) showing staining in lung tissue from MSC treated rats. (red = VE-Cadherin and DAPI staining nuclei -blue). Figure 6B: Staining of blood vessels in lungs (BVL = blood vessel lumen) for VE-Cadherin (red) and β-catenin (green) shows that in the LR group, HS and resuscitation compromise the continuity of the AJs (see white arrow). This continuity is preserved in MSC treated animals. Figure 6C: Similar findings are found for TJs staining of Occludin-1 (green) and Claudin-1 (red). MSCs preserve compromise of TJs in the lungs (see white arrows). Morphological changes (thickening) in AJs and TJs are noted in MSC treated lung vessels (long white arrows). Figures 6D–H show MSCs preserve PDGFRβ positive pericytes on lung microvasculature after HS. Sectioned lung tissue was stained for PDGFRβ (green) to identify pericytes or smooth muscle (SM) cells and vWF (red) to identify blood vessels. Figures 6E and 6F shows that HS and LR groups show diminished or compromised PDGFRβ staining (small white arrows), indicating decreased pericyte/SM cell coverage on microvasculature. This is increased above Sham ( Figure 6D ) in MSC treated animals ( Figure 6G -see large white arrow). Figure 6H shows that MSCs (red) contribute to some, but not all, of the increased PDGFRβ (green) staining found in treated lungs.

    Techniques Used: Staining

    Soluble factors play a role in the effects of IV MSCs in vivo. Figure 7 shows a working biological model of how MSCs may function biologically when delivered IV after HS. MSC attach to pulmonary vascular endothelial cells in the lungs where they produce soluble factor(s) that affect vascular stability in the lungs through modulation of AJs, TJs and checking inflammation. We hypothesize that the soluble factor(s) produced promote local and systemic vascular stability.
    Figure Legend Snippet: Soluble factors play a role in the effects of IV MSCs in vivo. Figure 7 shows a working biological model of how MSCs may function biologically when delivered IV after HS. MSC attach to pulmonary vascular endothelial cells in the lungs where they produce soluble factor(s) that affect vascular stability in the lungs through modulation of AJs, TJs and checking inflammation. We hypothesize that the soluble factor(s) produced promote local and systemic vascular stability.

    Techniques Used: In Vivo, Produced

    Conditioned media (CM) inhibits permeability and restores adherens junctions (AJs). Figure 1A , CM from MSCs and MSC-PEC co-cultures inhibits PEC permeability in vitro . A schematic depicting how CM is prepared is shown. MSCs and PECs are cultured separately and together in co-culture. CM is collected 24 hours later and used in a transwell assay of PEC permeability to 40 kD FITC-Dextran. Figure 1A shows that CM from both MSCs and MSC-PEC co-cultures inhibits permeability induced by VEGF-A (10 ng/ml). (*) signs indicate significance p
    Figure Legend Snippet: Conditioned media (CM) inhibits permeability and restores adherens junctions (AJs). Figure 1A , CM from MSCs and MSC-PEC co-cultures inhibits PEC permeability in vitro . A schematic depicting how CM is prepared is shown. MSCs and PECs are cultured separately and together in co-culture. CM is collected 24 hours later and used in a transwell assay of PEC permeability to 40 kD FITC-Dextran. Figure 1A shows that CM from both MSCs and MSC-PEC co-cultures inhibits permeability induced by VEGF-A (10 ng/ml). (*) signs indicate significance p

    Techniques Used: Permeability, In Vitro, Cell Culture, Co-Culture Assay, Transwell Assay

    In Vivo IV MSCs do not alter mean arterial pressure (MAP) in rat model of hemorrhagic shock and resuscitation. Figure 3A shows a schematic of the in vivo rat model. Animals were pre-instrumented three days prior to hemorrhage. Hemorrhage of fixed volume at a rate of 2 ml/100 g/10 minutes was performed. One hour later, resuscitation of Lactated Ringer's (LR) (3× shed blood) was administered. MSCs at a dose of 2×10 6 were administered with LR at 1 and 24 hours post hemorrhage. Blood was drawn at 0, 2 and 96 hours. Tissues were harvested on day four. Figures 3B and 3C show that hemorrhage volume and weight of rats is similar in all groups. Figure 3D shows that MAP drops respectively in all hemorrhaged groups and returns to normal by 2 hours post-hemorrhage. MSC administration does not affect MAP.
    Figure Legend Snippet: In Vivo IV MSCs do not alter mean arterial pressure (MAP) in rat model of hemorrhagic shock and resuscitation. Figure 3A shows a schematic of the in vivo rat model. Animals were pre-instrumented three days prior to hemorrhage. Hemorrhage of fixed volume at a rate of 2 ml/100 g/10 minutes was performed. One hour later, resuscitation of Lactated Ringer's (LR) (3× shed blood) was administered. MSCs at a dose of 2×10 6 were administered with LR at 1 and 24 hours post hemorrhage. Blood was drawn at 0, 2 and 96 hours. Tissues were harvested on day four. Figures 3B and 3C show that hemorrhage volume and weight of rats is similar in all groups. Figure 3D shows that MAP drops respectively in all hemorrhaged groups and returns to normal by 2 hours post-hemorrhage. MSC administration does not affect MAP.

    Techniques Used: In Vivo

    11) Product Images from "CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells"

    Article Title: CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells

    Journal: BioResearch Open Access

    doi: 10.1089/biores.2014.0029

    The effect of growth factor addition to unsorted MSC cocultures upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with MSCs culture conditions with supplemental growth factors (+GF) or without (–GF). SMCs cocultures (GF) and EOC monocultures (+GF) were used as controls. (B) EOC network morphology was observed over 14 days of coculture and quantified. Scale bar equals 200 μm. # Indicates p
    Figure Legend Snippet: The effect of growth factor addition to unsorted MSC cocultures upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with MSCs culture conditions with supplemental growth factors (+GF) or without (–GF). SMCs cocultures (GF) and EOC monocultures (+GF) were used as controls. (B) EOC network morphology was observed over 14 days of coculture and quantified. Scale bar equals 200 μm. # Indicates p

    Techniques Used: In Vitro, Transduction, Expressing

    Evaluation of the effect of CD45-expressing cells within MSC populations, upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with either CD45- MSCs, unsorted MSCs, or SMCs for observation over 14 days of coculture. Scale bar equals 200 μm. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis for total segment length, mean segment length, and number of branch points. * p
    Figure Legend Snippet: Evaluation of the effect of CD45-expressing cells within MSC populations, upon EOC network formation in vitro . (A) EOCs were transduced with tomato-expressing lentivirus and combined with either CD45- MSCs, unsorted MSCs, or SMCs for observation over 14 days of coculture. Scale bar equals 200 μm. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis for total segment length, mean segment length, and number of branch points. * p

    Techniques Used: Expressing, In Vitro, Transduction

    Comparison of mesenchymal stem cell (MSC) and smooth muscle cell (SMC) coculture upon endothelial outgrowth cell (EOC) network formation in vitro . Images are representative of three independent experiments. Scale bars equal 100 μm. (A) EOCs were transduced with tomato-expressing lentivirus and combined with either MSCs or SMCs for observation over 18 days of coculture. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis of the number of branch points (B) and mean segment length (C) . (D, E) EOCs in MSC cocultures underwent cell loss over 10 days of culture in contrast to SMCs as evidenced by the percentage of platelet endothelial cell adhesion molecule (PECAM)–positive cells, normalized to total cell number. Image area analyzed is 0.57 mm 2 with n =3 images analyzed per condition. *Indicates p
    Figure Legend Snippet: Comparison of mesenchymal stem cell (MSC) and smooth muscle cell (SMC) coculture upon endothelial outgrowth cell (EOC) network formation in vitro . Images are representative of three independent experiments. Scale bars equal 100 μm. (A) EOCs were transduced with tomato-expressing lentivirus and combined with either MSCs or SMCs for observation over 18 days of coculture. Changes in EOC network morphology between MSC and SMC coculture conditions over time were quantified through analysis of the number of branch points (B) and mean segment length (C) . (D, E) EOCs in MSC cocultures underwent cell loss over 10 days of culture in contrast to SMCs as evidenced by the percentage of platelet endothelial cell adhesion molecule (PECAM)–positive cells, normalized to total cell number. Image area analyzed is 0.57 mm 2 with n =3 images analyzed per condition. *Indicates p

    Techniques Used: In Vitro, Transduction, Expressing

    (A) Measured supernatant levels for thrombospondin-1 (TSP-1), interferon-gamma (IFN-γ), and interferon-alpha (IFN-α) in cocultures of EOCs with MSCs or SMCs during the first 18 days of culture. (B) Analysis of phagocytosis response within EOC and MSC cocultures during the first 10 days of culture; n =4 wells analyzed per condition. (C) Representative phagocytosis assay images depicting intracellular, fluorescein-labeled Escherichia coli within MSC cocultures during the first 10 days of coculture. Positive controls consisted of macrophages and negative controls consisted of experimental wells containing only Dulbecco's modified Eagle's medium. Scale bar equals 200 μm.
    Figure Legend Snippet: (A) Measured supernatant levels for thrombospondin-1 (TSP-1), interferon-gamma (IFN-γ), and interferon-alpha (IFN-α) in cocultures of EOCs with MSCs or SMCs during the first 18 days of culture. (B) Analysis of phagocytosis response within EOC and MSC cocultures during the first 10 days of culture; n =4 wells analyzed per condition. (C) Representative phagocytosis assay images depicting intracellular, fluorescein-labeled Escherichia coli within MSC cocultures during the first 10 days of coculture. Positive controls consisted of macrophages and negative controls consisted of experimental wells containing only Dulbecco's modified Eagle's medium. Scale bar equals 200 μm.

    Techniques Used: Phagocytosis Assay, Labeling, Modification

    12) Product Images from "Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators"

    Article Title: Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt418

    Role of human growth factors in induction of rat neural markers in various culture settings; qRT-PCR. (A) Effect of FGF2 inhibition on rat nestin. Rat neural cells were stimulated with SB623 cell-derived conditioned medium (25%), which increased the nestin expression. The presence of FGF2-neutralizing antibody (bFM1), but not FGF2-specific nonneutralizing antibody (bMF2), concentration-dependently inhibited the nestin increase. (B) Effect of human FGF1 inhibition on rat nestin. Rat neural cells were stimulated by MSCs (60 or 200 cells/well), MSC-CM (5%), or recombinant FGF1, or FGF2 (both at 5 ng/ml), which led to the induction of rat nestin. The application of the anti-human FGF1 neutralizing antibody decreased nestin mRNA levels induced by all stimuli but the recombinant FGF2. (C) Effect of BMP inhibition on rat GFAP induction. Rat GFAP was induced by coculturing rat neural cells with SB623 (200 cells/well). A BMP inhibitor noggin concentration-dependently decreased the GFAP induction. (D) Effect of HGF silencing in MSC on rat CNP induction. Rat CNP expression was stimulated by coculturing rat neural cells with MSC transfected with either HGF siRNA (siHGF) or control siRNA (siControl). A lower CNP induction was observed in cocultures with siHGF-transfectants.
    Figure Legend Snippet: Role of human growth factors in induction of rat neural markers in various culture settings; qRT-PCR. (A) Effect of FGF2 inhibition on rat nestin. Rat neural cells were stimulated with SB623 cell-derived conditioned medium (25%), which increased the nestin expression. The presence of FGF2-neutralizing antibody (bFM1), but not FGF2-specific nonneutralizing antibody (bMF2), concentration-dependently inhibited the nestin increase. (B) Effect of human FGF1 inhibition on rat nestin. Rat neural cells were stimulated by MSCs (60 or 200 cells/well), MSC-CM (5%), or recombinant FGF1, or FGF2 (both at 5 ng/ml), which led to the induction of rat nestin. The application of the anti-human FGF1 neutralizing antibody decreased nestin mRNA levels induced by all stimuli but the recombinant FGF2. (C) Effect of BMP inhibition on rat GFAP induction. Rat GFAP was induced by coculturing rat neural cells with SB623 (200 cells/well). A BMP inhibitor noggin concentration-dependently decreased the GFAP induction. (D) Effect of HGF silencing in MSC on rat CNP induction. Rat CNP expression was stimulated by coculturing rat neural cells with MSC transfected with either HGF siRNA (siHGF) or control siRNA (siControl). A lower CNP induction was observed in cocultures with siHGF-transfectants.

    Techniques Used: Quantitative RT-PCR, Inhibition, Derivative Assay, Expressing, Concentration Assay, Recombinant, Transfection

    Comparison of expression and activity of TGM2 in SB623 and MSC; its functional analysis in ECM using siRNA. (A) Expression levels of TGM2 normalized to GAP were determined by using qRT-PCR in SB623/MSC pairs from several donors. Levels in SB623 cells were expressed relative to levels in parental MSCs, which were set on 1. (B) TGM2-crosslinking activity was measured by amounts of biotinylated cadaverine incorporated into PLL in the presence of SB623 or MSC cell lysates. The activity was then normalized to the total protein, and expressed compared with the parental MSCs, where the values were set on 1. (C) TGM2 was detected in ECM of MSCs and SB623 by using immunoblotting. The TGM2 signal was quantified densitometrically and normalized to the total ECM protein per lane. The total ECM protein was assessed by using duplicated gel: the gel was stained for protein; photographed; and the density of corresponding lane minus background determined. (The whole blot and gel are shown in Additional file 6 : Figure S5). (D) Nestin expression was quantified by using qRT-PCR in rat neural cells grown on ECM produced by SB623 transfected with either siTGM2 or siControl. Nestin levels on siControl-ECM were set on 1. The graph represents means from three experiments; error bar represents standard error of mean; * P
    Figure Legend Snippet: Comparison of expression and activity of TGM2 in SB623 and MSC; its functional analysis in ECM using siRNA. (A) Expression levels of TGM2 normalized to GAP were determined by using qRT-PCR in SB623/MSC pairs from several donors. Levels in SB623 cells were expressed relative to levels in parental MSCs, which were set on 1. (B) TGM2-crosslinking activity was measured by amounts of biotinylated cadaverine incorporated into PLL in the presence of SB623 or MSC cell lysates. The activity was then normalized to the total protein, and expressed compared with the parental MSCs, where the values were set on 1. (C) TGM2 was detected in ECM of MSCs and SB623 by using immunoblotting. The TGM2 signal was quantified densitometrically and normalized to the total ECM protein per lane. The total ECM protein was assessed by using duplicated gel: the gel was stained for protein; photographed; and the density of corresponding lane minus background determined. (The whole blot and gel are shown in Additional file 6 : Figure S5). (D) Nestin expression was quantified by using qRT-PCR in rat neural cells grown on ECM produced by SB623 transfected with either siTGM2 or siControl. Nestin levels on siControl-ECM were set on 1. The graph represents means from three experiments; error bar represents standard error of mean; * P

    Techniques Used: Expressing, Activity Assay, Functional Assay, Quantitative RT-PCR, Staining, Produced, Transfection

    Comparison of neuropoietic activity of SB623 and MSCs in cocultures with rat embryonic neural cells. (A) Rat neural cells were grown in the presence or absence of MSCs or SB623 (rat/human cell ratio was 10:1) and immunostained for rat nestin (upper panel) and GFAP (middle panel) on day 5, or for CNP on day 12. Nuclei were stained with DAPI (B) An example of microplate neuropoiesis assay data: a comparison of rat neural differentiation marker induction in cocultures of rat cells with MSCs and SB623 from Donor A. Rat neural cells (5,000/well) were cocultured with 500, 250, and 125 cells/well of either MSCs or SB623; and expression of rat-specific nestin, GFAP, and CNP, and human-specific GAP was quantified by using qRT-PCR. Stimulation with 10% MSC-CM and no stimulation (“No add”) were used as positive control and background, respectively. Relative units correspond to standard samples used in qPCR run. Error bars represent SD of biologic duplicates. (C) Neuropoietic coefficients of MSCs and SB623 from Donor A were calculated based on data presented in (B) by first subtracting the background expression of a corresponding neural marker and then normalizing the expression of the neural marker to the human GAP for each number of human cells per well, followed by averaging normalized values. Error bars represent SD from three normalized values.
    Figure Legend Snippet: Comparison of neuropoietic activity of SB623 and MSCs in cocultures with rat embryonic neural cells. (A) Rat neural cells were grown in the presence or absence of MSCs or SB623 (rat/human cell ratio was 10:1) and immunostained for rat nestin (upper panel) and GFAP (middle panel) on day 5, or for CNP on day 12. Nuclei were stained with DAPI (B) An example of microplate neuropoiesis assay data: a comparison of rat neural differentiation marker induction in cocultures of rat cells with MSCs and SB623 from Donor A. Rat neural cells (5,000/well) were cocultured with 500, 250, and 125 cells/well of either MSCs or SB623; and expression of rat-specific nestin, GFAP, and CNP, and human-specific GAP was quantified by using qRT-PCR. Stimulation with 10% MSC-CM and no stimulation (“No add”) were used as positive control and background, respectively. Relative units correspond to standard samples used in qPCR run. Error bars represent SD of biologic duplicates. (C) Neuropoietic coefficients of MSCs and SB623 from Donor A were calculated based on data presented in (B) by first subtracting the background expression of a corresponding neural marker and then normalizing the expression of the neural marker to the human GAP for each number of human cells per well, followed by averaging normalized values. Error bars represent SD from three normalized values.

    Techniques Used: Activity Assay, Staining, Marker, Expressing, Quantitative RT-PCR, Positive Control, Real-time Polymerase Chain Reaction

    Comparison of presynaptic puncta formation in cocultures of rat embryonic neural cells with MSCs or SB623; immunostaining. (A) Immunostaining for VGLUT (day 7) and VGAT (day 11); rat/human cell ratio, 50:1. Bar, 50 μm. (B) Quantification of VGAT-immunoreactive puncta per neurite length (averaged from 10 microscopic fields, one to four neurites/field), day 11. (C) Typical distribution and size of VGAT-positive puncta in axonal processes in cocultures with MSCs (upper) or SB623 (lower), day 13. Neurites are outlined manually. Bar, 50 um.
    Figure Legend Snippet: Comparison of presynaptic puncta formation in cocultures of rat embryonic neural cells with MSCs or SB623; immunostaining. (A) Immunostaining for VGLUT (day 7) and VGAT (day 11); rat/human cell ratio, 50:1. Bar, 50 μm. (B) Quantification of VGAT-immunoreactive puncta per neurite length (averaged from 10 microscopic fields, one to four neurites/field), day 11. (C) Typical distribution and size of VGAT-positive puncta in axonal processes in cocultures with MSCs (upper) or SB623 (lower), day 13. Neurites are outlined manually. Bar, 50 um.

    Techniques Used: Immunostaining

    13) Product Images from "Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy"

    Article Title: Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028321

    Primed hMSCsPTX and SR4987PTX inhibit proliferation of different TC lines in vitro. In (A) is shown the kinetics of growth inhibition induced by serial dilutions of hMSCsPTX-CM on T98G, DU145 and of SR4987PTX-CM on B16 which was compared with activity of different concentrations of PTX on the same TCs (B). The CM addition produced a strong anti-proliferative effect on all TCs tested in a dose dependent manner: 1∶16 and 1∶4 dilutions IC 50 and IC 90 growth inhibition on all TC lines respectively, corresponding to the PTX concentrations necessary to obtain IC 50 and IC 90 on the different tumor cells as reported in small table insert in (B). Inhibition of TCs proliferation was also obtained by a direct co-culture assay. Primed hMSCsPTX mixed, at different ratios (1∶100–1∶10–1∶1 MSCs/TCs), with MOLT-4 (C), T98G (D), DU145 (E) showed dose dependent capacity to block TCs proliferation evaluated in a MTT test at 7 days expressed as percent of OD measured for TCs cultured in control medium alone (CTR) or in presence of not primed hMSCs. SR4987PTX behaved like hMSCsPTX. However, even not primed SR4987 per se showed some anti-proliferative capacity on B16 melanoma at 1∶1 ratio (F). The histograms report the mean ± SD of three experiments with the statistical significance as follows: * ( p
    Figure Legend Snippet: Primed hMSCsPTX and SR4987PTX inhibit proliferation of different TC lines in vitro. In (A) is shown the kinetics of growth inhibition induced by serial dilutions of hMSCsPTX-CM on T98G, DU145 and of SR4987PTX-CM on B16 which was compared with activity of different concentrations of PTX on the same TCs (B). The CM addition produced a strong anti-proliferative effect on all TCs tested in a dose dependent manner: 1∶16 and 1∶4 dilutions IC 50 and IC 90 growth inhibition on all TC lines respectively, corresponding to the PTX concentrations necessary to obtain IC 50 and IC 90 on the different tumor cells as reported in small table insert in (B). Inhibition of TCs proliferation was also obtained by a direct co-culture assay. Primed hMSCsPTX mixed, at different ratios (1∶100–1∶10–1∶1 MSCs/TCs), with MOLT-4 (C), T98G (D), DU145 (E) showed dose dependent capacity to block TCs proliferation evaluated in a MTT test at 7 days expressed as percent of OD measured for TCs cultured in control medium alone (CTR) or in presence of not primed hMSCs. SR4987PTX behaved like hMSCsPTX. However, even not primed SR4987 per se showed some anti-proliferative capacity on B16 melanoma at 1∶1 ratio (F). The histograms report the mean ± SD of three experiments with the statistical significance as follows: * ( p

    Techniques Used: In Vitro, Inhibition, Activity Assay, Produced, Co-culture Assay, Blocking Assay, MTT Assay, Cell Culture

    14) Product Images from "Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain"

    Article Title: Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201205056

    Durotaxis and MIIB polarization are disrupted by MIIA S1943D overexpression and are maximal for WT levels of MIIB expression and pS1943. (A) MSCs on gradient gels transfected with WT GFP-MIIA show a durotaxis index similar to nontransfected, WT cells (yellow band). The S1943A mutant shows a reduced durotaxis index, and the S1943D mutant shows no significant durotaxis above background. Data are means ± SEM for ≥12 cells. (B) The same transfected MSCs were immunostained for MIIB, which polarizes in GFP-MIIA cells on stiff matrix (34 kPa) but does not on soft matrix (1 kPa) similar to nontransfected, WT cells ( Fig. 1 ). The S1943A mutant shows a modest increase in MIIB polarization even on soft matrix, where these cells tend to spread more than any other cell ( Fig. S5 ), as is found on stiff matrix. The S1943D mutant shows no significant MIIB polarization on soft or stiff matrix, even though these cells tend to spread on stiff matrix more so than any other cell (Fig. S5). The blue band is the range of durotaxis index when there is no durotaxis per Fig. 3 C . (C) Summary of all durotaxis index and polarization data from WT, KD, and overexpression experiments shows that WT cells are optimized for durotaxis and polarization. For only the data point involving transfection with S1943D, the S1943D is considered equivalent to pS1943 and considered part of the percentage of pS1943 in the graph. The surface plot illustrates the sensitivity of durotaxis and MIIB polarization to both MIIB expression level and the percentage of MIIA that is phosphorylated at S1943 within the cell. Circles are red for the durotaxis index, whereas squares are black for rear/front fluorescence. Data are means ± SEM for ≥20 cells among three independent experiments. Bars, 50 µm.
    Figure Legend Snippet: Durotaxis and MIIB polarization are disrupted by MIIA S1943D overexpression and are maximal for WT levels of MIIB expression and pS1943. (A) MSCs on gradient gels transfected with WT GFP-MIIA show a durotaxis index similar to nontransfected, WT cells (yellow band). The S1943A mutant shows a reduced durotaxis index, and the S1943D mutant shows no significant durotaxis above background. Data are means ± SEM for ≥12 cells. (B) The same transfected MSCs were immunostained for MIIB, which polarizes in GFP-MIIA cells on stiff matrix (34 kPa) but does not on soft matrix (1 kPa) similar to nontransfected, WT cells ( Fig. 1 ). The S1943A mutant shows a modest increase in MIIB polarization even on soft matrix, where these cells tend to spread more than any other cell ( Fig. S5 ), as is found on stiff matrix. The S1943D mutant shows no significant MIIB polarization on soft or stiff matrix, even though these cells tend to spread on stiff matrix more so than any other cell (Fig. S5). The blue band is the range of durotaxis index when there is no durotaxis per Fig. 3 C . (C) Summary of all durotaxis index and polarization data from WT, KD, and overexpression experiments shows that WT cells are optimized for durotaxis and polarization. For only the data point involving transfection with S1943D, the S1943D is considered equivalent to pS1943 and considered part of the percentage of pS1943 in the graph. The surface plot illustrates the sensitivity of durotaxis and MIIB polarization to both MIIB expression level and the percentage of MIIA that is phosphorylated at S1943 within the cell. Circles are red for the durotaxis index, whereas squares are black for rear/front fluorescence. Data are means ± SEM for ≥20 cells among three independent experiments. Bars, 50 µm.

    Techniques Used: Over Expression, Expressing, Transfection, Mutagenesis, Fluorescence

    15) Product Images from "A Multi-Niche Microvascularized Human Bone-Marrow-on-a-Chip"

    Article Title: A Multi-Niche Microvascularized Human Bone-Marrow-on-a-Chip

    Journal: bioRxiv

    doi: 10.1101/2019.12.15.876813

    Schematic of human bone marrow-on-a-chip (hBM-on-a-chip). (A) The hBM-on-a-chip can recapitulate both the central perivascular BM niche (without OBs) and the vascularized endosteal BM niche (with OBs) that are found in the cavities of long bones. MSC = mesenchymal or marrow stromal cells, including pericytes; OB = osteoblasts and mineralized bone-like tissue layer; stromal cells = other cells of the BM stroma including CXCL12-abundant reticular cells (CAR), matured hematopoietic cells, and adipose cells. Note the area between vasculatures represent the central marrow region. FN = Fibronectin; LN = Laminin, Col-I and Col-IV = Collagen I and Collagen IV; OP = Osteopontin; Jag-1 = Jagged 1. (B) A 5-channel PDMS microfluidic device was fabricated using standard soft lithography techniques. MSCs are first differentiated for 21 days in the central channel of the device to form an endosteal layer, then HUVECs, MSCs, and HSPCs are loaded on top of the endosteal layer and vasculogenesis occurs over 5 days to form the hBM-on-a-chip.
    Figure Legend Snippet: Schematic of human bone marrow-on-a-chip (hBM-on-a-chip). (A) The hBM-on-a-chip can recapitulate both the central perivascular BM niche (without OBs) and the vascularized endosteal BM niche (with OBs) that are found in the cavities of long bones. MSC = mesenchymal or marrow stromal cells, including pericytes; OB = osteoblasts and mineralized bone-like tissue layer; stromal cells = other cells of the BM stroma including CXCL12-abundant reticular cells (CAR), matured hematopoietic cells, and adipose cells. Note the area between vasculatures represent the central marrow region. FN = Fibronectin; LN = Laminin, Col-I and Col-IV = Collagen I and Collagen IV; OP = Osteopontin; Jag-1 = Jagged 1. (B) A 5-channel PDMS microfluidic device was fabricated using standard soft lithography techniques. MSCs are first differentiated for 21 days in the central channel of the device to form an endosteal layer, then HUVECs, MSCs, and HSPCs are loaded on top of the endosteal layer and vasculogenesis occurs over 5 days to form the hBM-on-a-chip.

    Techniques Used: Chromatin Immunoprecipitation

    16) Product Images from "A Double Mechanism for the Mesenchymal Stem Cells' Positive Effect on Pancreatic Islets"

    Article Title: A Double Mechanism for the Mesenchymal Stem Cells' Positive Effect on Pancreatic Islets

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084309

    Insulin release after glucose stimulation. Each week (up to 4 weeks of culture) pancreatic islets cultured alone or directly co-cultured with MSCs, and MSCs cultured alone were exposed to different glucose concentrations in the culture medium (20 mM and 1,67 mM Glucose), and the insulin release after each change was measured by an ELISA assay specific for this hormone. Results are expressed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: Insulin release after glucose stimulation. Each week (up to 4 weeks of culture) pancreatic islets cultured alone or directly co-cultured with MSCs, and MSCs cultured alone were exposed to different glucose concentrations in the culture medium (20 mM and 1,67 mM Glucose), and the insulin release after each change was measured by an ELISA assay specific for this hormone. Results are expressed as mean ± SD of three independent experiments. * P

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NF-Kappa B"

    Article Title: Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NF-Kappa B

    Journal: Journal of tissue science & engineering

    doi: 10.4172/2157-7552.S3-001

    MSCs increase IL-10 production in U937-MSC co-culture Supernatants collected from MSC culture (150,000cells/well) alone, and U937 culture (3×10 5 /well and 7.5×10 5 /well) alone did not contain any IL-10, as quantified by cytokine bead assay for flow cytometry (BD Biosciences, San Jose, CA). Both co-culture groups (in ratios 1:2 and 1:5, MSCs:U937s) significantly increased IL-10 production in response to stimulation with LPS (5ng/mL).
    Figure Legend Snippet: MSCs increase IL-10 production in U937-MSC co-culture Supernatants collected from MSC culture (150,000cells/well) alone, and U937 culture (3×10 5 /well and 7.5×10 5 /well) alone did not contain any IL-10, as quantified by cytokine bead assay for flow cytometry (BD Biosciences, San Jose, CA). Both co-culture groups (in ratios 1:2 and 1:5, MSCs:U937s) significantly increased IL-10 production in response to stimulation with LPS (5ng/mL).

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry

    MSCs do not influence U937 leukocyte adhesion molecule expression A. Schematic of adhesion molecule assay: U937 leukocytes (500,000cells/well) in 6 well plates were cultured with or without MSCs (ratio of 1:5) for 24 hours. The cells were stimulated with PMA (200ng/mL) for 20 minutes. The cells were then labeled with fluorophore conjugated antibodies to CD62L, CD29, CD11b, and CD18. B. Image U937: Representative image of U937 leukocytes alone in culture. C. Image U937 and MSC co-culture: Representative image of U937 leukocytes in co-culture with MSCs. D. Mean Flourescent Intensity changes in all markers (n=4): There were no differences in adhesion molecule expression in U937 leukocytes after co-culture with MSCs and stimulation with PMA. Adhesion molecule expression was quantified by mean fluorescence intensity for four individual culture wells.
    Figure Legend Snippet: MSCs do not influence U937 leukocyte adhesion molecule expression A. Schematic of adhesion molecule assay: U937 leukocytes (500,000cells/well) in 6 well plates were cultured with or without MSCs (ratio of 1:5) for 24 hours. The cells were stimulated with PMA (200ng/mL) for 20 minutes. The cells were then labeled with fluorophore conjugated antibodies to CD62L, CD29, CD11b, and CD18. B. Image U937: Representative image of U937 leukocytes alone in culture. C. Image U937 and MSC co-culture: Representative image of U937 leukocytes in co-culture with MSCs. D. Mean Flourescent Intensity changes in all markers (n=4): There were no differences in adhesion molecule expression in U937 leukocytes after co-culture with MSCs and stimulation with PMA. Adhesion molecule expression was quantified by mean fluorescence intensity for four individual culture wells.

    Techniques Used: Expressing, Cell Culture, Labeling, Co-Culture Assay, Fluorescence

    18) Product Images from "Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain"

    Article Title: Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201205056

    Durotaxis and MIIB polarization are disrupted by MIIA S1943D overexpression and are maximal for WT levels of MIIB expression and pS1943. (A) MSCs on gradient gels transfected with WT GFP-MIIA show a durotaxis index similar to nontransfected, WT cells (yellow band). The S1943A mutant shows a reduced durotaxis index, and the S1943D mutant shows no significant durotaxis above background. Data are means ± SEM for ≥12 cells. (B) The same transfected MSCs were immunostained for MIIB, which polarizes in GFP-MIIA cells on stiff matrix (34 kPa) but does not on soft matrix (1 kPa) similar to nontransfected, WT cells ( Fig. 1 ). The S1943A mutant shows a modest increase in MIIB polarization even on soft matrix, where these cells tend to spread more than any other cell ( Fig. S5 ), as is found on stiff matrix. The S1943D mutant shows no significant MIIB polarization on soft or stiff matrix, even though these cells tend to spread on stiff matrix more so than any other cell (Fig. S5). The blue band is the range of durotaxis index when there is no durotaxis per Fig. 3 C . (C) Summary of all durotaxis index and polarization data from WT, KD, and overexpression experiments shows that WT cells are optimized for durotaxis and polarization. For only the data point involving transfection with S1943D, the S1943D is considered equivalent to pS1943 and considered part of the percentage of pS1943 in the graph. The surface plot illustrates the sensitivity of durotaxis and MIIB polarization to both MIIB expression level and the percentage of MIIA that is phosphorylated at S1943 within the cell. Circles are red for the durotaxis index, whereas squares are black for rear/front fluorescence. Data are means ± SEM for ≥20 cells among three independent experiments. Bars, 50 µm.
    Figure Legend Snippet: Durotaxis and MIIB polarization are disrupted by MIIA S1943D overexpression and are maximal for WT levels of MIIB expression and pS1943. (A) MSCs on gradient gels transfected with WT GFP-MIIA show a durotaxis index similar to nontransfected, WT cells (yellow band). The S1943A mutant shows a reduced durotaxis index, and the S1943D mutant shows no significant durotaxis above background. Data are means ± SEM for ≥12 cells. (B) The same transfected MSCs were immunostained for MIIB, which polarizes in GFP-MIIA cells on stiff matrix (34 kPa) but does not on soft matrix (1 kPa) similar to nontransfected, WT cells ( Fig. 1 ). The S1943A mutant shows a modest increase in MIIB polarization even on soft matrix, where these cells tend to spread more than any other cell ( Fig. S5 ), as is found on stiff matrix. The S1943D mutant shows no significant MIIB polarization on soft or stiff matrix, even though these cells tend to spread on stiff matrix more so than any other cell (Fig. S5). The blue band is the range of durotaxis index when there is no durotaxis per Fig. 3 C . (C) Summary of all durotaxis index and polarization data from WT, KD, and overexpression experiments shows that WT cells are optimized for durotaxis and polarization. For only the data point involving transfection with S1943D, the S1943D is considered equivalent to pS1943 and considered part of the percentage of pS1943 in the graph. The surface plot illustrates the sensitivity of durotaxis and MIIB polarization to both MIIB expression level and the percentage of MIIA that is phosphorylated at S1943 within the cell. Circles are red for the durotaxis index, whereas squares are black for rear/front fluorescence. Data are means ± SEM for ≥20 cells among three independent experiments. Bars, 50 µm.

    Techniques Used: Over Expression, Expressing, Transfection, Mutagenesis, Fluorescence

    19) Product Images from "Direct conversion of human fibroblasts into therapeutically active vascular wall-typical mesenchymal stem cells"

    Article Title: Direct conversion of human fibroblasts into therapeutically active vascular wall-typical mesenchymal stem cells

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-019-03358-0

    Regulated expression of HOXB7, HOXC6 and HOXC8. To address the question whether the reprogrammed state of the generated VW-MSCs is stable or not, a doxycycline-inducible retroviral expression vector (“all-in-one tet-on” system) for controllable expression of the HOX co-expression cassette was generated. a Scheme of the self-inactivating lentiviral vector for doxycycline-inducible expression of the coding sequences of HOXB7 , HOXC6 and HOXC8 separated by 2A esterase elements together with the gene encoding the reporter TURQUOISE2 fluorescent protein. b Primary human fibroblasts were transduced with the doxycycline inducible SIN vector encoding for the HOX code. Transduced cells (iHOX) were treated with doxycycline (0.2–0.5 µg/ml) 48 h after transduction, sorted for cyan fluorescence after expansion (additional 4–6 days) and then cultured in MSC medium. Mock-transduced fibroblasts either doxycycline-treated or not were used as control. Western blot analysis of total HOXB7, HOXC6 and HOXC8 protein expression as well as of Nestin (NES) marker protein expression was performed from whole cell lysates of HOX-transduced and control fibroblasts with or without doxycycline withdrawal for 4 days. Beta-actin (ACTIN) and alpha-tubulin (TUB) were included as loading controls. # Fibroblasts derived from (2) different healthy donors. No changes of the doxycycline-induced HOXB7, HOXC6 and HOXC8 protein expression levels were detectable 4 day after doxycycline withdrawal. c Relative amounts of transcripts of the introduced HOX genes were further determined by qRT-PCR 8 days after doxycycline withdrawal (biological replicates: n = 4 per group and gene; P by two-way ANOVA, followed by post hoc Tukey’s multiple comparisons test: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005; **** P ≤ 0.001). Relative transcript levels of analyzed genes were normalized to beta-actin mRNA (set as 1). No significant down-regulation of the doxycycline-induced HOXB7, HOXC6 and HOXC8 mRNA expression levels were detectable 8 day after doxycycline withdrawal, although the respective HOX expression levels trend to decline. d CFU Assay. The colony-forming potential of doxycycline-HOX-induced VW-MSCs as compared to primary fibroblasts was investigated by plating respective cells at low densities and subsequently culturing for 10 days in the presence or absence of doxycycline for the indicated days. Quantification of the surviving colonies revealed that 2–21 days after doxycycline-withdrawal generated VW-MSCs (iHOX) still possess colony-formation potential. e Verification of doxycycline-induced conversion into MSCs. FACS-purified, iHOX-transduced and control doxycycline-treated fibroblasts were differentiated into adipocytes, osteocytes and chondrocytes at the indicated time points after doxycycline removal. Differentiation was observed within 14 days after induction of differentiation (DM) as shown by Oil red staining (adipocytes), by histochemical staining for alkaline phosphatase (ALP) visualizing osteocytes, or by immunocytochemistry for cartilage-specific collagen type II expressions (chondrocytes). Representative photographs are shown. Magnification × 400. As control, respective cells were cultured in normal growth media (NGM). Of note, 14–21 days after doxycycline-withdrawal generated VW-MSCs (iHOX) still possessed trilineage differentiation potential
    Figure Legend Snippet: Regulated expression of HOXB7, HOXC6 and HOXC8. To address the question whether the reprogrammed state of the generated VW-MSCs is stable or not, a doxycycline-inducible retroviral expression vector (“all-in-one tet-on” system) for controllable expression of the HOX co-expression cassette was generated. a Scheme of the self-inactivating lentiviral vector for doxycycline-inducible expression of the coding sequences of HOXB7 , HOXC6 and HOXC8 separated by 2A esterase elements together with the gene encoding the reporter TURQUOISE2 fluorescent protein. b Primary human fibroblasts were transduced with the doxycycline inducible SIN vector encoding for the HOX code. Transduced cells (iHOX) were treated with doxycycline (0.2–0.5 µg/ml) 48 h after transduction, sorted for cyan fluorescence after expansion (additional 4–6 days) and then cultured in MSC medium. Mock-transduced fibroblasts either doxycycline-treated or not were used as control. Western blot analysis of total HOXB7, HOXC6 and HOXC8 protein expression as well as of Nestin (NES) marker protein expression was performed from whole cell lysates of HOX-transduced and control fibroblasts with or without doxycycline withdrawal for 4 days. Beta-actin (ACTIN) and alpha-tubulin (TUB) were included as loading controls. # Fibroblasts derived from (2) different healthy donors. No changes of the doxycycline-induced HOXB7, HOXC6 and HOXC8 protein expression levels were detectable 4 day after doxycycline withdrawal. c Relative amounts of transcripts of the introduced HOX genes were further determined by qRT-PCR 8 days after doxycycline withdrawal (biological replicates: n = 4 per group and gene; P by two-way ANOVA, followed by post hoc Tukey’s multiple comparisons test: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005; **** P ≤ 0.001). Relative transcript levels of analyzed genes were normalized to beta-actin mRNA (set as 1). No significant down-regulation of the doxycycline-induced HOXB7, HOXC6 and HOXC8 mRNA expression levels were detectable 8 day after doxycycline withdrawal, although the respective HOX expression levels trend to decline. d CFU Assay. The colony-forming potential of doxycycline-HOX-induced VW-MSCs as compared to primary fibroblasts was investigated by plating respective cells at low densities and subsequently culturing for 10 days in the presence or absence of doxycycline for the indicated days. Quantification of the surviving colonies revealed that 2–21 days after doxycycline-withdrawal generated VW-MSCs (iHOX) still possess colony-formation potential. e Verification of doxycycline-induced conversion into MSCs. FACS-purified, iHOX-transduced and control doxycycline-treated fibroblasts were differentiated into adipocytes, osteocytes and chondrocytes at the indicated time points after doxycycline removal. Differentiation was observed within 14 days after induction of differentiation (DM) as shown by Oil red staining (adipocytes), by histochemical staining for alkaline phosphatase (ALP) visualizing osteocytes, or by immunocytochemistry for cartilage-specific collagen type II expressions (chondrocytes). Representative photographs are shown. Magnification × 400. As control, respective cells were cultured in normal growth media (NGM). Of note, 14–21 days after doxycycline-withdrawal generated VW-MSCs (iHOX) still possessed trilineage differentiation potential

    Techniques Used: Expressing, Generated, Plasmid Preparation, Transduction, Fluorescence, Cell Culture, Western Blot, Marker, Derivative Assay, Quantitative RT-PCR, Colony-forming Unit Assay, FACS, Purification, Staining, Immunocytochemistry

    20) Product Images from "Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine"

    Article Title: Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0936-8

    Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. ** p
    Figure Legend Snippet: Alkaline phosphatase and adenosine deaminase activity and function blocking. a Alkaline phosphatase (ALP) and b adenosine deaminase (ADA) activity in different cell types. Individual biological replicates depicted as dots. c Effects induced by adding ALP inhibitor levamisole or ADA in platelet–cell cocultures. Data normalized against control activated by TRAP-6 without cells (dotted line at value 1). w/o n = 4–11, BM-MSCs n = 9, LA-MSCs n = 7–12, CB-MSCs n = 5–9, HUVECs n = 6 biological replicates; HeLa cells n = 3, adenosine n = 4. ** p

    Techniques Used: Activity Assay, Blocking Assay, ALP Assay

    Effect of conditioned medium from different cell types on platelet activation. Platelets incubated with either cell culture or conditioned medium (CM) and then stimulated by TRAP-6. Platelet activation measured by flow cytometry, assessing expression of CD62P, CD63 and PAC-1 binding. a Bone marrow (BM)-MSCs. b Lipoaspirate (LA)-MSCs. c Cord blood (CB)-MSCs. d Human umbilical vein endothelial cells (HUVECs). e HeLa tumor cells. Scale indicates relative activation marker expression of CM compared to culture medium. n = 3 biological replicates; HeLa cells n = 1, note different y axis for HeLa cells. * p
    Figure Legend Snippet: Effect of conditioned medium from different cell types on platelet activation. Platelets incubated with either cell culture or conditioned medium (CM) and then stimulated by TRAP-6. Platelet activation measured by flow cytometry, assessing expression of CD62P, CD63 and PAC-1 binding. a Bone marrow (BM)-MSCs. b Lipoaspirate (LA)-MSCs. c Cord blood (CB)-MSCs. d Human umbilical vein endothelial cells (HUVECs). e HeLa tumor cells. Scale indicates relative activation marker expression of CM compared to culture medium. n = 3 biological replicates; HeLa cells n = 1, note different y axis for HeLa cells. * p

    Techniques Used: Activation Assay, Incubation, Cell Culture, Flow Cytometry, Cytometry, Expressing, Binding Assay, Marker

    Expression and ectonucleotidase activity of CD39, CD73 and A2AR. a Cells stained with respective antibodies and MFI values determined by flow cytometry. BM-MSCs n = 3, LA-MSCs n = 7, CB-MSCs n = 5, HUVECs, n = 6 each biological replicates; HeLa n = 3 technical replicates; Plt unst, Plt stim each n = 4 biological replicates. * p
    Figure Legend Snippet: Expression and ectonucleotidase activity of CD39, CD73 and A2AR. a Cells stained with respective antibodies and MFI values determined by flow cytometry. BM-MSCs n = 3, LA-MSCs n = 7, CB-MSCs n = 5, HUVECs, n = 6 each biological replicates; HeLa n = 3 technical replicates; Plt unst, Plt stim each n = 4 biological replicates. * p

    Techniques Used: Expressing, Activity Assay, Staining, Flow Cytometry, Cytometry

    21) Product Images from "From the Cover: S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells"

    Article Title: From the Cover: S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells

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

    doi: 10.1073/pnas.1220185110

    Down-regulation of PDGFRα in GSNOR −/− MSCs. ( A–C ) Representative FACS analysis depicting absence of VEGFR2 (
    Figure Legend Snippet: Down-regulation of PDGFRα in GSNOR −/− MSCs. ( A–C ) Representative FACS analysis depicting absence of VEGFR2 (

    Techniques Used: FACS

    MSCs from GSNOR −/− mice exhibit reduced endothelial differentiation and impaired blood vessel formation in vivo. ( A and B ) Matrigel plug (2 wk after injection) containing GFP + MSCs from WT and GSNOR −/− . ( B ) H E staining with blood vessel formation indicated by the black arrows ( C ) MSCs from GSNOR −/− mice following isolectin (red) and GFP (green) staining shows colocalization (orange) and exhibit diminished potential to differentiate into endothelial cells than WT MSCs. ( D ) Quantification of the number of GFP + blood vessels containing autofluorescent red blood cells [white arrows, see (A)]. ( E ) Quantification of blood vessel formation 2 wk after injection. * P
    Figure Legend Snippet: MSCs from GSNOR −/− mice exhibit reduced endothelial differentiation and impaired blood vessel formation in vivo. ( A and B ) Matrigel plug (2 wk after injection) containing GFP + MSCs from WT and GSNOR −/− . ( B ) H E staining with blood vessel formation indicated by the black arrows ( C ) MSCs from GSNOR −/− mice following isolectin (red) and GFP (green) staining shows colocalization (orange) and exhibit diminished potential to differentiate into endothelial cells than WT MSCs. ( D ) Quantification of the number of GFP + blood vessels containing autofluorescent red blood cells [white arrows, see (A)]. ( E ) Quantification of blood vessel formation 2 wk after injection. * P

    Techniques Used: Mouse Assay, In Vivo, Injection, Staining

    22) Product Images from "Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces"

    Article Title: Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2010.0431

    Differences in gene expression and adipogenic and osteogenic differentiation between 3 odontogenic progenitors, DF, DP, and PDL, compared with MSCs. (A) Differences in gene expression between DF, PDL, DP, and MSCs as revealed by reverse transcriptase–polymerase
    Figure Legend Snippet: Differences in gene expression and adipogenic and osteogenic differentiation between 3 odontogenic progenitors, DF, DP, and PDL, compared with MSCs. (A) Differences in gene expression between DF, PDL, DP, and MSCs as revealed by reverse transcriptase–polymerase

    Techniques Used: Expressing

    23) Product Images from "Characterization of Autologous Mesenchymal Stem Cell-Derived Neural Progenitors as a Feasible Source of Stem Cells for Central Nervous System Applications in Multiple Sclerosis"

    Article Title: Characterization of Autologous Mesenchymal Stem Cell-Derived Neural Progenitors as a Feasible Source of Stem Cells for Central Nervous System Applications in Multiple Sclerosis

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2012-0015

    MSC-NPs lose mesodermal differentiation capacity. (A): Alizarin red staining of calcium deposition after osteogenic differentiation demonstrating limited osteogenic differentiation by MSC-NPs compared with MSCs. (B): Oil Red O staining of accumulated
    Figure Legend Snippet: MSC-NPs lose mesodermal differentiation capacity. (A): Alizarin red staining of calcium deposition after osteogenic differentiation demonstrating limited osteogenic differentiation by MSC-NPs compared with MSCs. (B): Oil Red O staining of accumulated

    Techniques Used: Staining

    Immunoregulatory properties of MSC-NPs and MSCs are similar. (A): Proliferation of phytohemagglutinin-stimulated CFSE-labeled CD4 + T cells (white histogram) is reduced in the presence of cocultured MSC-NPs (black filled histogram) as indicated by a right
    Figure Legend Snippet: Immunoregulatory properties of MSC-NPs and MSCs are similar. (A): Proliferation of phytohemagglutinin-stimulated CFSE-labeled CD4 + T cells (white histogram) is reduced in the presence of cocultured MSC-NPs (black filled histogram) as indicated by a right

    Techniques Used: Labeling

    Expression of neural and mesenchymal protein markers in MSC-NPs. (A): Immunocytochemistry demonstrating qualitative changes in Nestin, NF-M, GFAP, CXCR4, SMA, and CD90 in MSC-NPs compared with MSCs. A representative experiment using MS patient-derived
    Figure Legend Snippet: Expression of neural and mesenchymal protein markers in MSC-NPs. (A): Immunocytochemistry demonstrating qualitative changes in Nestin, NF-M, GFAP, CXCR4, SMA, and CD90 in MSC-NPs compared with MSCs. A representative experiment using MS patient-derived

    Techniques Used: Expressing, Immunocytochemistry, Mass Spectrometry, Derivative Assay

    Characterization of MSC-NP morphology and gene expression. (A): Spindle-shaped morphology of human MSCs viewed by light microscopy at a magnification of ×10. (B): Spherical morphology of MSC-NPs after 3 weeks of neural induction. The cells were
    Figure Legend Snippet: Characterization of MSC-NP morphology and gene expression. (A): Spindle-shaped morphology of human MSCs viewed by light microscopy at a magnification of ×10. (B): Spherical morphology of MSC-NPs after 3 weeks of neural induction. The cells were

    Techniques Used: Expressing, Light Microscopy

    24) Product Images from "Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo"

    Article Title: Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo

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

    doi: 10.1073/pnas.1601911113

    Growth factor secretion by mesenchymal stromal cells is enhanced by coculture with H5N1 influenza virus-infected alveolar epithelial cells and contributes to reduction of AFC and APP impairment. Secretion of ( A ) Ang1 and ( B ) KGF by MSCs was increased
    Figure Legend Snippet: Growth factor secretion by mesenchymal stromal cells is enhanced by coculture with H5N1 influenza virus-infected alveolar epithelial cells and contributes to reduction of AFC and APP impairment. Secretion of ( A ) Ang1 and ( B ) KGF by MSCs was increased

    Techniques Used: Infection

    The effect of MSCs on H5N1-impaired AFC ( Left ) and APP ( Right ) is dependent on the number of MSC used for coculture. Alveolar epithelial cell monolayers on the apical chamber of transwell culture inserts were infected with H5N1 (A/HongKong/483/97) influenza
    Figure Legend Snippet: The effect of MSCs on H5N1-impaired AFC ( Left ) and APP ( Right ) is dependent on the number of MSC used for coculture. Alveolar epithelial cell monolayers on the apical chamber of transwell culture inserts were infected with H5N1 (A/HongKong/483/97) influenza

    Techniques Used: Infection

    H5N1 influenza virus infection of alveolar epithelium induces greater cytokine and chemokine expression and lower sodium and chloride transporter expression than does H1N1 virus infection whereas coculture with MSCs prevents or reduces the H5N1 effects.
    Figure Legend Snippet: H5N1 influenza virus infection of alveolar epithelium induces greater cytokine and chemokine expression and lower sodium and chloride transporter expression than does H1N1 virus infection whereas coculture with MSCs prevents or reduces the H5N1 effects.

    Techniques Used: Infection, Expressing

    25) Product Images from "Validation of the 1, 4‐butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications, et al. Validation of the 1, 4‐butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications"

    Article Title: Validation of the 1, 4‐butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications, et al. Validation of the 1, 4‐butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications

    Journal: Bioengineering & Translational Medicine

    doi: 10.1002/btm2.10192

    MSCs chondrogenic differentiation in b‐TPUe bioprinted scaffolds. Chondrogenic differentiation was evaluated in MSCs cultured in monolayer, b‐TPUe scaffolds (CTL), and b‐TPUe scaffolds under differentiation conditions (Diff) after 21 days in culture. (a) RT‐PCR analysis of chondrogenic key markers. (b) GAGs quantification. (c) Type II collagen quantification. (d–f) SEM representative images of MSCs growing in b‐TPUe bioprinted scaffolds at day 21 (** p
    Figure Legend Snippet: MSCs chondrogenic differentiation in b‐TPUe bioprinted scaffolds. Chondrogenic differentiation was evaluated in MSCs cultured in monolayer, b‐TPUe scaffolds (CTL), and b‐TPUe scaffolds under differentiation conditions (Diff) after 21 days in culture. (a) RT‐PCR analysis of chondrogenic key markers. (b) GAGs quantification. (c) Type II collagen quantification. (d–f) SEM representative images of MSCs growing in b‐TPUe bioprinted scaffolds at day 21 (** p

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a"

    Article Title: Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2016-0020

    Sequential treatment with BMP‐2 followed by Wnt5a induces sustained expression of chondrocyte matrix markers under chondrogenic conditions. (A): Schematic shows treatment conditions for in vitro chondrogenic differentiation of H9‐MSCs. (B–E): Expression data based on quantitative polymerase chain reaction analyses of cartilage matrix genes in treatment groups from differentiating H9‐derived mesenchymal stem cell (MSC) pellets cultured for 5 days in 100 ng/ml BMP‐2 followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a): ratio of COL2A1:COL1A1 (B) , ACAN (C) , COL11A1 (D) , and expression of COL9A1 (E) . Values greater than 1.0 represent a fold‐change increase in gene expression, and those less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p
    Figure Legend Snippet: Sequential treatment with BMP‐2 followed by Wnt5a induces sustained expression of chondrocyte matrix markers under chondrogenic conditions. (A): Schematic shows treatment conditions for in vitro chondrogenic differentiation of H9‐MSCs. (B–E): Expression data based on quantitative polymerase chain reaction analyses of cartilage matrix genes in treatment groups from differentiating H9‐derived mesenchymal stem cell (MSC) pellets cultured for 5 days in 100 ng/ml BMP‐2 followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a): ratio of COL2A1:COL1A1 (B) , ACAN (C) , COL11A1 (D) , and expression of COL9A1 (E) . Values greater than 1.0 represent a fold‐change increase in gene expression, and those less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p

    Techniques Used: Expressing, In Vitro, Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture

    Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.
    Figure Legend Snippet: Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.

    Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, Polymerase Chain Reaction, Staining, Cell Culture, ALP Assay, FACS

    Regeneration of permanent cartilage‐like tissue by implanted H9‐derived mesenchymal stem cells (MSCs) pretreated with sequential BMP‐2 and Wnt5a. Quantitative reverse‐transcriptase polymerase chain reaction of preamplified tissue scraped from the defected regions on prepared slides of paraffin‐embedded rat knees 4 and 8 weeks after surgery is shown. Human‐specific expressions of the early chondrogenic gene marker COL2A1 and articular chondrocyte matrix markers COL11A1 , ACAN , and PRG4 were detected in only in tissue scrapings from animals receiving the untreated human H9‐derived MSCs, and the human H9‐derived MSCs pretreated for 14 days (2 days, BMP‐2, 12 days, Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p
    Figure Legend Snippet: Regeneration of permanent cartilage‐like tissue by implanted H9‐derived mesenchymal stem cells (MSCs) pretreated with sequential BMP‐2 and Wnt5a. Quantitative reverse‐transcriptase polymerase chain reaction of preamplified tissue scraped from the defected regions on prepared slides of paraffin‐embedded rat knees 4 and 8 weeks after surgery is shown. Human‐specific expressions of the early chondrogenic gene marker COL2A1 and articular chondrocyte matrix markers COL11A1 , ACAN , and PRG4 were detected in only in tissue scrapings from animals receiving the untreated human H9‐derived MSCs, and the human H9‐derived MSCs pretreated for 14 days (2 days, BMP‐2, 12 days, Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p

    Techniques Used: Derivative Assay, Polymerase Chain Reaction, Marker, Expressing

    Wnt5a induced chondrogenic gene expression and limited expression of the hypertrophic chondrocyte markers in H9‐derived MSC pellets. Comparative expression of early chondro‐progenitor genes SOX9 (A) and COL2A1 (B) and the expression of mature chondrocyte markers of hypertrophy COL10A1 (C) and ALP (D) in H9‐derived MSC pellets cultured for 14 days with or without 100 ng/ml BMP‐2 or 50 ng/ml Wnt5a. Values greater than 1.0 represent a fold‐change increase in gene expression and less than a 1.0 relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p
    Figure Legend Snippet: Wnt5a induced chondrogenic gene expression and limited expression of the hypertrophic chondrocyte markers in H9‐derived MSC pellets. Comparative expression of early chondro‐progenitor genes SOX9 (A) and COL2A1 (B) and the expression of mature chondrocyte markers of hypertrophy COL10A1 (C) and ALP (D) in H9‐derived MSC pellets cultured for 14 days with or without 100 ng/ml BMP‐2 or 50 ng/ml Wnt5a. Values greater than 1.0 represent a fold‐change increase in gene expression and less than a 1.0 relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p

    Techniques Used: Expressing, Derivative Assay, ALP Assay, Cell Culture

    Wnt5a suppressed BMP‐2‐induced markers of chondrocyte hypertrophy. Comparative expression of mature chondrocyte markers of hypertrophy COL10A1 (A) and ALP (B) in H9‐derived mesenchymal stem cells (MSCs) cultured for 5 days in 100 ng/ml BMP‐2, followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p
    Figure Legend Snippet: Wnt5a suppressed BMP‐2‐induced markers of chondrocyte hypertrophy. Comparative expression of mature chondrocyte markers of hypertrophy COL10A1 (A) and ALP (B) in H9‐derived mesenchymal stem cells (MSCs) cultured for 5 days in 100 ng/ml BMP‐2, followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p

    Techniques Used: Expressing, ALP Assay, Derivative Assay, Cell Culture

    27) Product Images from "Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling"

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.06.015

    Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p
    Figure Legend Snippet: Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p

    Techniques Used: Microarray, Expressing, Immunofluorescence, Staining, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p
    Figure Legend Snippet: TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p

    Techniques Used: Inhibition, Imaging, Cell Culture, Fluorescence, Expressing

    TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p
    Figure Legend Snippet: TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p

    Techniques Used: Cell Culture, Expressing

    28) Product Images from "Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling"

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.06.015

    Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p
    Figure Legend Snippet: Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p

    Techniques Used: Microarray, Expressing, Immunofluorescence, Staining, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p
    Figure Legend Snippet: TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p

    Techniques Used: Inhibition, Imaging, Cell Culture, Fluorescence, Expressing

    TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p
    Figure Legend Snippet: TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p

    Techniques Used: Cell Culture, Expressing

    29) Product Images from "Human Mesenchymal Stem Cells Inhibit Endothelial Proliferation and Angiogenesis via Cell-Cell Contact Through Modulation of the VE-Cadherin/?-Catenin Signaling Pathway"

    Article Title: Human Mesenchymal Stem Cells Inhibit Endothelial Proliferation and Angiogenesis via Cell-Cell Contact Through Modulation of the VE-Cadherin/?-Catenin Signaling Pathway

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2012.0165

    Coculture of MSCs in contact with ECs inhibits vascular network formation in vitro. (A) ECs cultured in contact with MSCs for 2 days were separated by MACs using negative selection of CD44-positive cells and then seeded into matrigel. (B) After 6 h
    Figure Legend Snippet: Coculture of MSCs in contact with ECs inhibits vascular network formation in vitro. (A) ECs cultured in contact with MSCs for 2 days were separated by MACs using negative selection of CD44-positive cells and then seeded into matrigel. (B) After 6 h

    Techniques Used: In Vitro, Cell Culture, Magnetic Cell Separation, Selection

    Mesenchymal stem cells (MSCs) in contact with endothelial cells (ECs) inhibit EC proliferation and S-phase cell cycle progression in a dose-dependent fashion. (A) Antibody-based separation of MSCs from ECs using an antibody against CD44 (which is present
    Figure Legend Snippet: Mesenchymal stem cells (MSCs) in contact with endothelial cells (ECs) inhibit EC proliferation and S-phase cell cycle progression in a dose-dependent fashion. (A) Antibody-based separation of MSCs from ECs using an antibody against CD44 (which is present

    Techniques Used:

    Coculture of MSCs in contact with ECs alters the release of a soluble factor(s) that affects EC angiogenic capacity. (A) ECs cultured in contact with MSCs were placed in a transwell above ECs seeded into matrigel. After 6 h in matrigel, the ECs
    Figure Legend Snippet: Coculture of MSCs in contact with ECs alters the release of a soluble factor(s) that affects EC angiogenic capacity. (A) ECs cultured in contact with MSCs were placed in a transwell above ECs seeded into matrigel. After 6 h in matrigel, the ECs

    Techniques Used: Cell Culture

    Activation of the Wnt pathway abrogates EC adherens junctions and inhibits vascular network formation in matrigel. (A) ECs cocultured with Wnt3a-transfected MSCs display diminished localization of β-catenin at the membrane, which results in a
    Figure Legend Snippet: Activation of the Wnt pathway abrogates EC adherens junctions and inhibits vascular network formation in matrigel. (A) ECs cocultured with Wnt3a-transfected MSCs display diminished localization of β-catenin at the membrane, which results in a

    Techniques Used: Activation Assay, Transfection

    30) Product Images from "Microvessel Network Formation and Interactions with Pancreatic Islets in Three-Dimensional Chip Cultures"

    Article Title: Microvessel Network Formation and Interactions with Pancreatic Islets in Three-Dimensional Chip Cultures

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2019.0186

    Effect of cell types and culture medium on microvascular network formation in 2D and 3D. (A) Representative images of vascular networks formed by HUVECs and iPSC-ECFC cocultured with MSCs or human lung fibroblasts in different culture media. Images were taken after 3 and 6 days of culture. Scale bars, 500 μm. (B) Representative images of vascular networks formed by HUVECs alone, HUVECs+human lung fibroblasts, HUVECs+adipose-derived MSCs, and iPSC-ECFC+adipose-derived MSCs in a fibrin gel. Images were taken after 5 days of culture. Scale bars, 200 μm. 2D, two-dimensional; 3D, three-dimensional; HUVECs, human umbilical vein endothelial cells; iPSC-ECFC, induced pluripotent stem cell-derived endothelial colony-forming cell; MSCs, mesenchymal stem cells.
    Figure Legend Snippet: Effect of cell types and culture medium on microvascular network formation in 2D and 3D. (A) Representative images of vascular networks formed by HUVECs and iPSC-ECFC cocultured with MSCs or human lung fibroblasts in different culture media. Images were taken after 3 and 6 days of culture. Scale bars, 500 μm. (B) Representative images of vascular networks formed by HUVECs alone, HUVECs+human lung fibroblasts, HUVECs+adipose-derived MSCs, and iPSC-ECFC+adipose-derived MSCs in a fibrin gel. Images were taken after 5 days of culture. Scale bars, 200 μm. 2D, two-dimensional; 3D, three-dimensional; HUVECs, human umbilical vein endothelial cells; iPSC-ECFC, induced pluripotent stem cell-derived endothelial colony-forming cell; MSCs, mesenchymal stem cells.

    Techniques Used: Derivative Assay

    Microvascular network recruitment to islets. (A) Progression of microvascular network formation around a rat islet day by day. Cells were evenly dispersed in the gel immediately after seeding, and gradually formed a mostly interconnected network by day 5, assembling around the islets. Two million RFP-HUVECs were cocultured with AT-MSCs at a 5:1 ratio in a fibrin gel with integrated GFP rat islets. Scale bars, 200 μm. (B) . Scale bar, 100 μm. (C) Distribution of vascular network coverage immediately around islets at days 1 and 4. For analysis of network area coverage around islets, the regions that were analyzed contained the islet and the area extending 100 μm from the islet boundary. FIJI was used to automatically threshold and analyze the images. The vascular density around the islets increased significantly from day 1 (12.3% ± 0.5%) to day 4 (20.0% ± 0.9%) ( p -value
    Figure Legend Snippet: Microvascular network recruitment to islets. (A) Progression of microvascular network formation around a rat islet day by day. Cells were evenly dispersed in the gel immediately after seeding, and gradually formed a mostly interconnected network by day 5, assembling around the islets. Two million RFP-HUVECs were cocultured with AT-MSCs at a 5:1 ratio in a fibrin gel with integrated GFP rat islets. Scale bars, 200 μm. (B) . Scale bar, 100 μm. (C) Distribution of vascular network coverage immediately around islets at days 1 and 4. For analysis of network area coverage around islets, the regions that were analyzed contained the islet and the area extending 100 μm from the islet boundary. FIJI was used to automatically threshold and analyze the images. The vascular density around the islets increased significantly from day 1 (12.3% ± 0.5%) to day 4 (20.0% ± 0.9%) ( p -value

    Techniques Used:

    Microvascular networks form perfusable lumens. (A) Orthogonal projections of z -stack images of RFP-HUVECs cultured with AT-MSCs in a fibrin gel for 5 days. Images were composed from 0.9 μm serial confocal images (72 slices) through the z -plane of the cells. Left panel shows stacked xy projection, and the side and bottom panels show yz and xz projections. Yellow crosshairs indicate intersection of yz and yx planes. Rightmost panel shows single slice from stack at z -depth as indicated by the x and y axis crosshairs . (B) Fluorescent microparticles added to the medium channel enter the microvascular network, demonstrating that the engineered networks are perfusable. Scale bars, 25 μm. AT, adipose tissue.
    Figure Legend Snippet: Microvascular networks form perfusable lumens. (A) Orthogonal projections of z -stack images of RFP-HUVECs cultured with AT-MSCs in a fibrin gel for 5 days. Images were composed from 0.9 μm serial confocal images (72 slices) through the z -plane of the cells. Left panel shows stacked xy projection, and the side and bottom panels show yz and xz projections. Yellow crosshairs indicate intersection of yz and yx planes. Rightmost panel shows single slice from stack at z -depth as indicated by the x and y axis crosshairs . (B) Fluorescent microparticles added to the medium channel enter the microvascular network, demonstrating that the engineered networks are perfusable. Scale bars, 25 μm. AT, adipose tissue.

    Techniques Used: Cell Culture

    31) Product Images from "Human Mesenchymal Stem Cells Inhibit Vascular Permeability by Modulating Vascular Endothelial Cadherin/?-Catenin Signaling"

    Article Title: Human Mesenchymal Stem Cells Inhibit Vascular Permeability by Modulating Vascular Endothelial Cadherin/?-Catenin Signaling

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2010.0013

    Conditioned medium from MSC + HUVEC coculture recapitulates the effects of MSCs on EC permeability. (A) Experimental schematic of transwell cocultures with MSCs ± HUVECs to determine if a secreted factor(s) from
    Figure Legend Snippet: Conditioned medium from MSC + HUVEC coculture recapitulates the effects of MSCs on EC permeability. (A) Experimental schematic of transwell cocultures with MSCs ± HUVECs to determine if a secreted factor(s) from

    Techniques Used: Permeability

    32) Product Images from "Bone tissue engineering via human induced pluripotent, umbilical cord and bone marrow mesenchymal stem cells in rat cranium"

    Article Title: Bone tissue engineering via human induced pluripotent, umbilical cord and bone marrow mesenchymal stem cells in rat cranium

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2015.02.011

    Immunohistochemical staining for human nuclear antigen 12 w after implantation. hiPS-MSCs (A), hUCMSCs (B), hBMSCs (C), and CPC control (D). The inset in (A) is the negative control without primary antibody incubation. Positive staining (marked by arrows)
    Figure Legend Snippet: Immunohistochemical staining for human nuclear antigen 12 w after implantation. hiPS-MSCs (A), hUCMSCs (B), hBMSCs (C), and CPC control (D). The inset in (A) is the negative control without primary antibody incubation. Positive staining (marked by arrows)

    Techniques Used: Immunohistochemistry, Staining, Negative Control, Incubation

    Histomorphometry analysis of new bone area fraction (A), and new blood vessel density (B). The new bone area and new blood vessel density of hiPSC-MSCs, hUCMSCs and hBMSCs were higher than CPC control (p
    Figure Legend Snippet: Histomorphometry analysis of new bone area fraction (A), and new blood vessel density (B). The new bone area and new blood vessel density of hiPSC-MSCs, hUCMSCs and hBMSCs were higher than CPC control (p

    Techniques Used:

    High magnification images of new bone within the dotted rectangles in , respectively. (A) hiPSC-MSCs, (B) hBMSCs. Macropores from mannitol dissolution were filled with new bone. Osteoblasts lined on the surface of new bone. Osteocytes resided
    Figure Legend Snippet: High magnification images of new bone within the dotted rectangles in , respectively. (A) hiPSC-MSCs, (B) hBMSCs. Macropores from mannitol dissolution were filled with new bone. Osteoblasts lined on the surface of new bone. Osteocytes resided

    Techniques Used:

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

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

    Journal: Aging Cell

    doi: 10.1111/acel.13152

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

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

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

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

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

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

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

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

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

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

    34) Product Images from "Osteogenic Differentiation of Stem Cells Alters Vitamin D Receptor Expression"

    Article Title: Osteogenic Differentiation of Stem Cells Alters Vitamin D Receptor Expression

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2011.0411

    Western blot of vitamin D receptors in human mesenchymal stem cells. MSCs were cultured with (GM) or osteogenic medium (OST) for 14 days as described. Western blots against VDR and PDIA3 were performed. (A) Western blots of MSC lysates against VDR, PDIA3,
    Figure Legend Snippet: Western blot of vitamin D receptors in human mesenchymal stem cells. MSCs were cultured with (GM) or osteogenic medium (OST) for 14 days as described. Western blots against VDR and PDIA3 were performed. (A) Western blots of MSC lysates against VDR, PDIA3,

    Techniques Used: Western Blot, Cell Culture

    35) Product Images from "Diabetes Relief in Mice by Glucose-Sensing Insulin-Secreting Human α-Cells"

    Article Title: Diabetes Relief in Mice by Glucose-Sensing Insulin-Secreting Human α-Cells

    Journal: Nature

    doi: 10.1038/s41586-019-0942-8

    Reaggregation of dispersed purified human β-cells. ( a ) Pure β-cells were labeled with GFP and traced in 3 different culture conditions: in monolayer (“single β”), β-cell-only aggregation (“β”), or β-cell aggregation with stromal cells including HUVECs and MSCs (“β+HM”). Live imaging at indicated days (middle panels) and immunofluorescence at day 7 (right panels) show β-cell-only pseudoislets were self-organized by Day 5, whereas β+HM aggregates were constituted in only 1 day. β-cells in β+HM pseudoislets located at the periphery, while HM cells formed the core of the aggregates (red and blue, respectively). ( b ) To determine the optimal number of β-cells per pseudoislet, GFP + transduced β-cells were seeded on aggregation-plate-wells at the indicated densities. 7 days after culture, aggregates were harvested and analyzed. Aggregates were uniform in size. Pseudoislet size correlated with the number of cells seeded per well. It was reported that human islet cell aggregates with a diameter 100—150 μm, consisting of 1000 cells, show a comparable function to native islets 24 ; we thus decided to perform reaggregation experiments at 1000 β-cells/pseudoislet (1000 β-cells, 129.6±3.1 μm). β-cell aggregates with HM were also analyzed. n = 8 pseudoislets for 500 β-cells, n = 8 pseudoislets for 1000 β-cells, n = 9 pseudoislets for 2000 β-cells, n = 8 pseudoislets for 3000 β-cells, and n = 52 pseudoislets for 1000 β-cells + 400 HUVECs + 100 MSCs. ( c ) Immunofluorescence at indicated time-points in β-cell pseudoislets and β-cell+HM pseudoislets. ( d ) TUNEL staining (green) showed rare apoptotic cells (0.8%) in β-cell aggregates after 7 day-culture. ( e ) qPCR analyses of INSULIN and PDX1 expression in monolayer and aggregated β-cells showing higher expression of β-cell markers in reaggregated β-cells. Data are expressed as fold-change relative to the value in single β-cells. * p = 0.022 in qPCR for INS , * p = 0.026 in qPCR for PDX1 , Mann-Whitney test, two-tailed. n = 6 donor samples. ( f ) ELISA measurements of static glucose-stimulated human insulin release at 3 mM (Low) and 20 mM (Hi) glucose showing glucose-responsive C-peptide secretion in both β and β+HM aggregates, but not in single β-cells. **** p
    Figure Legend Snippet: Reaggregation of dispersed purified human β-cells. ( a ) Pure β-cells were labeled with GFP and traced in 3 different culture conditions: in monolayer (“single β”), β-cell-only aggregation (“β”), or β-cell aggregation with stromal cells including HUVECs and MSCs (“β+HM”). Live imaging at indicated days (middle panels) and immunofluorescence at day 7 (right panels) show β-cell-only pseudoislets were self-organized by Day 5, whereas β+HM aggregates were constituted in only 1 day. β-cells in β+HM pseudoislets located at the periphery, while HM cells formed the core of the aggregates (red and blue, respectively). ( b ) To determine the optimal number of β-cells per pseudoislet, GFP + transduced β-cells were seeded on aggregation-plate-wells at the indicated densities. 7 days after culture, aggregates were harvested and analyzed. Aggregates were uniform in size. Pseudoislet size correlated with the number of cells seeded per well. It was reported that human islet cell aggregates with a diameter 100—150 μm, consisting of 1000 cells, show a comparable function to native islets 24 ; we thus decided to perform reaggregation experiments at 1000 β-cells/pseudoislet (1000 β-cells, 129.6±3.1 μm). β-cell aggregates with HM were also analyzed. n = 8 pseudoislets for 500 β-cells, n = 8 pseudoislets for 1000 β-cells, n = 9 pseudoislets for 2000 β-cells, n = 8 pseudoislets for 3000 β-cells, and n = 52 pseudoislets for 1000 β-cells + 400 HUVECs + 100 MSCs. ( c ) Immunofluorescence at indicated time-points in β-cell pseudoislets and β-cell+HM pseudoislets. ( d ) TUNEL staining (green) showed rare apoptotic cells (0.8%) in β-cell aggregates after 7 day-culture. ( e ) qPCR analyses of INSULIN and PDX1 expression in monolayer and aggregated β-cells showing higher expression of β-cell markers in reaggregated β-cells. Data are expressed as fold-change relative to the value in single β-cells. * p = 0.022 in qPCR for INS , * p = 0.026 in qPCR for PDX1 , Mann-Whitney test, two-tailed. n = 6 donor samples. ( f ) ELISA measurements of static glucose-stimulated human insulin release at 3 mM (Low) and 20 mM (Hi) glucose showing glucose-responsive C-peptide secretion in both β and β+HM aggregates, but not in single β-cells. **** p

    Techniques Used: Purification, Labeling, Imaging, Immunofluorescence, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY, Two Tailed Test, Enzyme-linked Immunosorbent Assay

    36) Product Images from "TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties"

    Article Title: TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.596831

    Expression of TNFR2 is crucial for mesenchymal stem cells (MSCs) to support endothelial cell (EC) angiogenic function. To evaluate the impact of MSCs on human umbilical vein endothelial cell (HUVEC) angiogenic capacity, wild-type (WT) or TNFR2 knockout (KO)-MSCs (P2 and P3) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) medium. After 2 days, CM were taken, filtered, and added to HUVECs on Matrigel. HUVECs cultured in EBM2 basal medium were used as negative control, and HUVECs cultured in EGM2 complete medium were used as positive control. (A) Pictures were taken every 2 h, using objectives 4× and 10× of the inverted microscope in phase-contrast mode. Images were further analyzed to evaluate (B) the tube length and (C) the network structural complexity. Results are collected from three independent experiments ( n = 10). CM, conditioned media.
    Figure Legend Snippet: Expression of TNFR2 is crucial for mesenchymal stem cells (MSCs) to support endothelial cell (EC) angiogenic function. To evaluate the impact of MSCs on human umbilical vein endothelial cell (HUVEC) angiogenic capacity, wild-type (WT) or TNFR2 knockout (KO)-MSCs (P2 and P3) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) medium. After 2 days, CM were taken, filtered, and added to HUVECs on Matrigel. HUVECs cultured in EBM2 basal medium were used as negative control, and HUVECs cultured in EGM2 complete medium were used as positive control. (A) Pictures were taken every 2 h, using objectives 4× and 10× of the inverted microscope in phase-contrast mode. Images were further analyzed to evaluate (B) the tube length and (C) the network structural complexity. Results are collected from three independent experiments ( n = 10). CM, conditioned media.

    Techniques Used: Expressing, Knock-Out, Cell Culture, Modification, Negative Control, Positive Control, Inverted Microscopy

    37) Product Images from "Human Mesenchymal Stem Cells Inhibit Endothelial Proliferation and Angiogenesis via Cell-Cell Contact Through Modulation of the VE-Cadherin/?-Catenin Signaling Pathway"

    Article Title: Human Mesenchymal Stem Cells Inhibit Endothelial Proliferation and Angiogenesis via Cell-Cell Contact Through Modulation of the VE-Cadherin/?-Catenin Signaling Pathway

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2012.0165

    MSCs enhance EC stability through direct EC-MSC interactions and the release of soluble factors. Working biological model showing how MSCs inhibit EC proliferation, migration, and sprouting via release paracrine factors and direct effects following EC-MSC
    Figure Legend Snippet: MSCs enhance EC stability through direct EC-MSC interactions and the release of soluble factors. Working biological model showing how MSCs inhibit EC proliferation, migration, and sprouting via release paracrine factors and direct effects following EC-MSC

    Techniques Used: Migration

    38) Product Images from "Metabolism as an early predictor of DPSCs aging"

    Article Title: Metabolism as an early predictor of DPSCs aging

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37489-4

    The gene expression of DPSCs compared to MSCs, Fibroblasts and hESCs. ( a ) Principal component analysis (PCA) of DPSCs, MSCs and hESCs showed that DPSCs and MSCs grouped together. ( b )Principal component analysis (PCA) of DPSCs with skin fibroblasts and MSCs from literature showed that DPSCs have distinct expression profiles from foreskin fibroblasts. ( c ) Volcano plot of genes differentially expressed in MSCs/DPSCs vs. hESCs. ( d ) Volcano plot of genes differentially expressed in MSCs/DPSCs compared to fibroblasts. ( e ) Volcano plot of genes differentially expressed in DPSCs vs. MSCs.
    Figure Legend Snippet: The gene expression of DPSCs compared to MSCs, Fibroblasts and hESCs. ( a ) Principal component analysis (PCA) of DPSCs, MSCs and hESCs showed that DPSCs and MSCs grouped together. ( b )Principal component analysis (PCA) of DPSCs with skin fibroblasts and MSCs from literature showed that DPSCs have distinct expression profiles from foreskin fibroblasts. ( c ) Volcano plot of genes differentially expressed in MSCs/DPSCs vs. hESCs. ( d ) Volcano plot of genes differentially expressed in MSCs/DPSCs compared to fibroblasts. ( e ) Volcano plot of genes differentially expressed in DPSCs vs. MSCs.

    Techniques Used: Expressing

    BARX1 gene as a new specific marker for DPSCs. ( a – c ) Immunofluorescent staining of Barx1 in skin fibroblasts, adult DPSCs (DPSC 29), and deciduous DPSCs (DPSC 292) at low power magnification. ( d – f ) Showing the nuclear localization of Barx1 transcription factor at high power magnification. Exposure settings and laser intensity of the confocal microscope were adjusted and normalized for fibroblasts, and same settings were used for the DPSCs. ( g ) Quantitative PCR reveals absence of BARX1 gene in BM-MSCs and in skin fibroblasts. Fold change normalized to DPSC Lonza. Significance was determined by unpaired Student’s t-test; n = 3–6 per cell line; **p
    Figure Legend Snippet: BARX1 gene as a new specific marker for DPSCs. ( a – c ) Immunofluorescent staining of Barx1 in skin fibroblasts, adult DPSCs (DPSC 29), and deciduous DPSCs (DPSC 292) at low power magnification. ( d – f ) Showing the nuclear localization of Barx1 transcription factor at high power magnification. Exposure settings and laser intensity of the confocal microscope were adjusted and normalized for fibroblasts, and same settings were used for the DPSCs. ( g ) Quantitative PCR reveals absence of BARX1 gene in BM-MSCs and in skin fibroblasts. Fold change normalized to DPSC Lonza. Significance was determined by unpaired Student’s t-test; n = 3–6 per cell line; **p

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

    39) Product Images from "Testing the potency of anti-TNF-α and anti-IL-1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells"

    Article Title: Testing the potency of anti-TNF-α and anti-IL-1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells

    Journal: Biotechnology progress

    doi: 10.1002/btpr.2629

    a) Gene expression profiles following the addition of inflammatory mediators TNF-α, IL-1β or MCM h.s. working solution and anti-inflammatory biological drugs adalimumab (ADA), infliximab (IFX), etanercept (ETA) and anakinra (ANA) Blue and green dots represent values obtained in microspheroid chondral tissues made of MSCs and OACs (3 donors), respectively. Statistically significant changes, i.e. log 2 (RQ) ≥ 1 and ≤ −1 have been outlined; median values of all groups are also shown. b) Radar graphs representing anti-TNF-α neutralization efficacies of ADA ( blue ), IFX ( red ) and ETA ( green ). Mean RQ values of three biological samples are shown for OAC- and MSC-derived microspheroids. Value 0 represents total inhibition of gene expression.
    Figure Legend Snippet: a) Gene expression profiles following the addition of inflammatory mediators TNF-α, IL-1β or MCM h.s. working solution and anti-inflammatory biological drugs adalimumab (ADA), infliximab (IFX), etanercept (ETA) and anakinra (ANA) Blue and green dots represent values obtained in microspheroid chondral tissues made of MSCs and OACs (3 donors), respectively. Statistically significant changes, i.e. log 2 (RQ) ≥ 1 and ≤ −1 have been outlined; median values of all groups are also shown. b) Radar graphs representing anti-TNF-α neutralization efficacies of ADA ( blue ), IFX ( red ) and ETA ( green ). Mean RQ values of three biological samples are shown for OAC- and MSC-derived microspheroids. Value 0 represents total inhibition of gene expression.

    Techniques Used: Expressing, Neutralization, Derivative Assay, Inhibition

    Related Articles

    Isolation:

    Article Title: Soft extracellular matrix enhances inflammatory activation of mesenchymal stromal cells to induce monocyte production and trafficking
    Article Snippet: Since monocytes generated from the BM contribute to the pool of tissue macrophages in various disease conditions including tissue damage and fibrosis , the ability to systematically control monocyte turnover, as exemplified by our approach, may help inform therapeutic strategies for these diseases. .. MSC isolation and expansion MSCs were derived by plastic adherence (“passage 0”) of mononucleated cells from human BM aspirate donors (Lonza). .. After plastic adherence for 3 days, nonadherent cells were washed out, and adherent cells were cultured at 37°C in 5% CO2 in the MSC medium: α-minimal essential medium (αMEM) supplemented with 20% fetal bovine serum (Atlanta Biologicals), 1% penicillin/streptomycin (P/S; Thermo Fisher Scientific), and 1% GlutaMAX (Thermo Fisher Scientific).

    Derivative Assay:

    Article Title: Soft extracellular matrix enhances inflammatory activation of mesenchymal stromal cells to induce monocyte production and trafficking
    Article Snippet: Since monocytes generated from the BM contribute to the pool of tissue macrophages in various disease conditions including tissue damage and fibrosis , the ability to systematically control monocyte turnover, as exemplified by our approach, may help inform therapeutic strategies for these diseases. .. MSC isolation and expansion MSCs were derived by plastic adherence (“passage 0”) of mononucleated cells from human BM aspirate donors (Lonza). .. After plastic adherence for 3 days, nonadherent cells were washed out, and adherent cells were cultured at 37°C in 5% CO2 in the MSC medium: α-minimal essential medium (αMEM) supplemented with 20% fetal bovine serum (Atlanta Biologicals), 1% penicillin/streptomycin (P/S; Thermo Fisher Scientific), and 1% GlutaMAX (Thermo Fisher Scientific).

    Cell Culture:

    Article Title: TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties
    Article Snippet: Pictures were taken every 2 h, till 24 h, using a camera (Nikon D3100, Japan) installed on an inverted microscope (Nikon ECLIPSE T5100) with 4× and 10× objectives in phase-contrast mode. .. For evaluating the impact of MSCs on HUVEC angiogenesis, 5 × 105 WT or TNFR2 KO-MSCs were seeded in 75 cm2 flasks and cultured for 48 h, in EBM2 medium containing 5% FBS (Lonza) and 1% P/S/N (Gibco). ..

    Article Title: Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation
    Article Snippet: MC3T3-E1 cells (Riken BRC Cell Bank, Tsukuba, Japan) were cultured in α -minimum essential medium (α -MEM) with 10% FBS and 1% penicillin/streptomycin. .. Rat primary mesenchymal stem cells (Lonza, Walkersville, MD, USA) were cultured using an R-MSCGMBullet Kit (Lonza). .. These cells were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts
    Article Snippet: One day prior to transfection, MSCs from the 4th passage were grown to a confluency of 85% and 1.5×106 cells were counted. .. The plasmid bearing pre-miR-210 (vector with the target gene) and two control plasmids, one containing pmaxGFP (an empty vector) and the other containing Scramble (vector with a sequence that does not belong to any known miRNA), were transfected separately into MSCs using a Human MSC Nucleofector kit (Lonza, USA) and U23 program. .. Following 48 hours of transfection, the expression of GFP was detected in the cells under a fluorescent microscope, indicating the percentage of transfected cells.

    Sequencing:

    Article Title: Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts
    Article Snippet: One day prior to transfection, MSCs from the 4th passage were grown to a confluency of 85% and 1.5×106 cells were counted. .. The plasmid bearing pre-miR-210 (vector with the target gene) and two control plasmids, one containing pmaxGFP (an empty vector) and the other containing Scramble (vector with a sequence that does not belong to any known miRNA), were transfected separately into MSCs using a Human MSC Nucleofector kit (Lonza, USA) and U23 program. .. Following 48 hours of transfection, the expression of GFP was detected in the cells under a fluorescent microscope, indicating the percentage of transfected cells.

    Transfection:

    Article Title: Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts
    Article Snippet: One day prior to transfection, MSCs from the 4th passage were grown to a confluency of 85% and 1.5×106 cells were counted. .. The plasmid bearing pre-miR-210 (vector with the target gene) and two control plasmids, one containing pmaxGFP (an empty vector) and the other containing Scramble (vector with a sequence that does not belong to any known miRNA), were transfected separately into MSCs using a Human MSC Nucleofector kit (Lonza, USA) and U23 program. .. Following 48 hours of transfection, the expression of GFP was detected in the cells under a fluorescent microscope, indicating the percentage of transfected cells.

    Staining:

    Article Title: The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair
    Article Snippet: .. Following the protocol described in the MSC Adipogenesis Kit (Chemicon SCR020), we found that MSCs differentiated into adipocytes bearing oil-positive signals (positive AdipoRedTM assay reagent staining, Lonza). .. NRLM-MSCs expressed CD29, CD44, CD73, CD105, and CD166 but did express the hematopoietic markers CD34 and CD45, similar to the CD marker expression of MSCs maintained in standard MSC growth medium (MSCGM-MSCs).

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    Lonza mesenchymal stem cell growth medium
    Effects of increased IL-8 levels on hematopoietic <t>stem</t> cells. a Conditioned media was collected from 24-h cultures of healthy hBMSCs grown in <t>mesenchymal</t> stem <t>cell</t> <t>growth</t> <t>medium.</t> Subsequently, 2.5 × 10 6 hHSCs were cultured for 24 h in conditioned media in the presence n = 12) and absence ( n = 12) of IL-8 (100 ng/mL). The numbers of total, CD34+, and CD34+/CD45- cells were significantly higher in the hHSCs cultured in the presence of IL-8, compared to those in cells cultured without IL-8 ( p = 0.014, p = 0.020, and p = 0.039, respectively). b The relative expression of CXCR2 , mTOR , and c- MYC increased at 1 h after IL-8 treatment. The expression of CXCR2 returned to normal after 6 h of IL-8 treatment, and the expression of mTOR gradually decreased at 6 and 24 h after IL-8 treatment. In the case of c -MYC , the increased expression lasted up to 24 h. Each experiment was repeated thrice. Note: *** p
    Mesenchymal Stem Cell Growth Medium, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza human msc
    Human <t>MSC</t> grown in Plate-1 ( a, d ), Plate-2 ( b, e ), and controls ( b, f ) respectively (from left to right) imaged after 7 days of culture. In the top row shown are cells cultured using EC medium and in the bottom row are cells cultured using HMSC medium. Cells were stained using antibody for <t>CD31</t> (green) and imaged using both phasecontrast and fluorescence. Scale bar in all images depicts 100 μm (color figure online)
    Human Msc, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of increased IL-8 levels on hematopoietic stem cells. a Conditioned media was collected from 24-h cultures of healthy hBMSCs grown in mesenchymal stem cell growth medium. Subsequently, 2.5 × 10 6 hHSCs were cultured for 24 h in conditioned media in the presence n = 12) and absence ( n = 12) of IL-8 (100 ng/mL). The numbers of total, CD34+, and CD34+/CD45- cells were significantly higher in the hHSCs cultured in the presence of IL-8, compared to those in cells cultured without IL-8 ( p = 0.014, p = 0.020, and p = 0.039, respectively). b The relative expression of CXCR2 , mTOR , and c- MYC increased at 1 h after IL-8 treatment. The expression of CXCR2 returned to normal after 6 h of IL-8 treatment, and the expression of mTOR gradually decreased at 6 and 24 h after IL-8 treatment. In the case of c -MYC , the increased expression lasted up to 24 h. Each experiment was repeated thrice. Note: *** p

    Journal: BMC Cancer

    Article Title: Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

    doi: 10.1186/s12885-020-07102-x

    Figure Lengend Snippet: Effects of increased IL-8 levels on hematopoietic stem cells. a Conditioned media was collected from 24-h cultures of healthy hBMSCs grown in mesenchymal stem cell growth medium. Subsequently, 2.5 × 10 6 hHSCs were cultured for 24 h in conditioned media in the presence n = 12) and absence ( n = 12) of IL-8 (100 ng/mL). The numbers of total, CD34+, and CD34+/CD45- cells were significantly higher in the hHSCs cultured in the presence of IL-8, compared to those in cells cultured without IL-8 ( p = 0.014, p = 0.020, and p = 0.039, respectively). b The relative expression of CXCR2 , mTOR , and c- MYC increased at 1 h after IL-8 treatment. The expression of CXCR2 returned to normal after 6 h of IL-8 treatment, and the expression of mTOR gradually decreased at 6 and 24 h after IL-8 treatment. In the case of c -MYC , the increased expression lasted up to 24 h. Each experiment was repeated thrice. Note: *** p

    Article Snippet: Mononuclear cells (MNCs) were separated using Ficoll-Paque™ Plus medium (GE Healthcare Life Sciences, Seoul, South Korea); the remaining cells were cultured in mesenchymal stem cell growth medium (Lonza, Walkersville, MD, USA).

    Techniques: Cell Culture, Expressing

    Requirement for SOX9, TGF‐β, and high density in chondrogenic miRNA expression. (A–D): Mesenchymal stem cells (MSCs) were transfected for 3 days with SOX9‐targeting or nontargeting control siRNA prior to chondrogenic differentiation for 7 days in hanging transwell inserts. RNA was extracted at Days 0, 3, and 7, and the indicated gene expression assessed by real‐time RT‐PCR. (A): SOX9 expression normalized to 18S. (B): Day 3 chondrogenesis disc overlaid on transwell membrane. (C): Days 3 and 7 chondrogenesis gene expression normalized to 18S. (D): Days 3 and 7 chondrogenesis miRNA expression following SOX9 depletion. Expression is normalized to U6 and presented as a percentage of nontargeting control levels. (E–H): MSCs were cultured in chondrogenic differentiation medium with or without TGF‐β3 for 7 days in hanging transwell inserts to form a cartilaginous disc or in monolayer at low cell density. (E): Day 3 chondrogenesis disc overlaid on transwell membrane. (F): Days 3 and 7 chondrogenesis gene expression normalized to 18S. (G, H): Days 3 and 7 chondrogenesis miRNA expression following (G) TGF‐β3 removal or (H) monolayer culture. Expression is normalized to U6 and presented as a percentage of control levels. Values are the mean ± SEM of data pooled from three separate MSC donors. *, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

    doi: 10.1002/stem.2093

    Figure Lengend Snippet: Requirement for SOX9, TGF‐β, and high density in chondrogenic miRNA expression. (A–D): Mesenchymal stem cells (MSCs) were transfected for 3 days with SOX9‐targeting or nontargeting control siRNA prior to chondrogenic differentiation for 7 days in hanging transwell inserts. RNA was extracted at Days 0, 3, and 7, and the indicated gene expression assessed by real‐time RT‐PCR. (A): SOX9 expression normalized to 18S. (B): Day 3 chondrogenesis disc overlaid on transwell membrane. (C): Days 3 and 7 chondrogenesis gene expression normalized to 18S. (D): Days 3 and 7 chondrogenesis miRNA expression following SOX9 depletion. Expression is normalized to U6 and presented as a percentage of nontargeting control levels. (E–H): MSCs were cultured in chondrogenic differentiation medium with or without TGF‐β3 for 7 days in hanging transwell inserts to form a cartilaginous disc or in monolayer at low cell density. (E): Day 3 chondrogenesis disc overlaid on transwell membrane. (F): Days 3 and 7 chondrogenesis gene expression normalized to 18S. (G, H): Days 3 and 7 chondrogenesis miRNA expression following (G) TGF‐β3 removal or (H) monolayer culture. Expression is normalized to U6 and presented as a percentage of control levels. Values are the mean ± SEM of data pooled from three separate MSC donors. *, p

    Article Snippet: Human Bone Marrow Stem Cell Culture Human bone marrow MSCs (from seven donors, 18–25 years of age) were isolated from human bone marrow mononuclear cells (Lonza Biosciences, Berkshire, U.K.) by adherence for more than 24 hours to tissue culture plastic and were expanded in monolayer culture in mesenchymal stem cell growth medium (Lonza Biosciences) supplemented with 5 ng/ml fibroblast growth factor‐2 (R & D Systems, Abingdon, U.K.).

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

    Profile of miRNA expression during chondrogenesis. MSCs were cultured in chondrogenic differentiation medium for 14 days in hanging transwell inserts to form a cartilaginous disc. RNA was extracted at the indicated time points between Day 0 and Day 14. (A): Heatmap representation of more than twofold significantly regulated miRNA expression assessed by microarray from one MSC donor. (B): Expression of selected miRNAs in data pooled three from MSC donors by real‐time RT‐PCR. (C): Venn diagram of common miRNAs regulated in human MSC and mouse ATDC5 chondrogenesis. miRNAs in red or blue are upregulated or downregulated, respectively, in both models of chondrogenesis. Abbreviation: MSC, mesenchymal stem cell.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

    doi: 10.1002/stem.2093

    Figure Lengend Snippet: Profile of miRNA expression during chondrogenesis. MSCs were cultured in chondrogenic differentiation medium for 14 days in hanging transwell inserts to form a cartilaginous disc. RNA was extracted at the indicated time points between Day 0 and Day 14. (A): Heatmap representation of more than twofold significantly regulated miRNA expression assessed by microarray from one MSC donor. (B): Expression of selected miRNAs in data pooled three from MSC donors by real‐time RT‐PCR. (C): Venn diagram of common miRNAs regulated in human MSC and mouse ATDC5 chondrogenesis. miRNAs in red or blue are upregulated or downregulated, respectively, in both models of chondrogenesis. Abbreviation: MSC, mesenchymal stem cell.

    Article Snippet: Human Bone Marrow Stem Cell Culture Human bone marrow MSCs (from seven donors, 18–25 years of age) were isolated from human bone marrow mononuclear cells (Lonza Biosciences, Berkshire, U.K.) by adherence for more than 24 hours to tissue culture plastic and were expanded in monolayer culture in mesenchymal stem cell growth medium (Lonza Biosciences) supplemented with 5 ng/ml fibroblast growth factor‐2 (R & D Systems, Abingdon, U.K.).

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

    Expression of the immunological molecule IDO. (A) RT-PCR analysis of IDO gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Relative gene expression levels of IDO normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) Left: Western blot analysis of IDO protein expression stimulated with IFN-γ/TNF-α (+) or unstimulated (–) after 48 h. An internal standard of IDO ranging from 25 to 200 ng was included. One representative blot of three independent experiments is shown. Right: Comparison of IDO protein expression in ng per 1 μg total protein of three IFN-γ/TNF-α stimulated batches.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Expression of the immunological molecule IDO. (A) RT-PCR analysis of IDO gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Relative gene expression levels of IDO normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) Left: Western blot analysis of IDO protein expression stimulated with IFN-γ/TNF-α (+) or unstimulated (–) after 48 h. An internal standard of IDO ranging from 25 to 200 ng was included. One representative blot of three independent experiments is shown. Right: Comparison of IDO protein expression in ng per 1 μg total protein of three IFN-γ/TNF-α stimulated batches.

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Western Blot

    Examination of cytoskeleton morphology. (A) Images of ventral, dorsal stress fibers and transverse arcs compared to the influence of MSCGM™ or MSCBM™ with 8% HPL. Higher magnification of the individual fibers is shown in the lower left corner. (B) Left: Total numbers of stress fibers. Right: Total number of stress fibers split in ventral, dorsal stress fibers and transverse arcs. (C) Left: Number of fibers per cells, Mann-Whitney test. Right: Median of the length per cell, unpaired t -test. The same 20 cells per media were compared in (B,C) . ** p

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Examination of cytoskeleton morphology. (A) Images of ventral, dorsal stress fibers and transverse arcs compared to the influence of MSCGM™ or MSCBM™ with 8% HPL. Higher magnification of the individual fibers is shown in the lower left corner. (B) Left: Total numbers of stress fibers. Right: Total number of stress fibers split in ventral, dorsal stress fibers and transverse arcs. (C) Left: Number of fibers per cells, Mann-Whitney test. Right: Median of the length per cell, unpaired t -test. The same 20 cells per media were compared in (B,C) . ** p

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: MANN-WHITNEY

    Examination of elasticity. (A) Left: Topographic images of membrane structures from MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL from passage 1 and 3 determined by atomic force microscopy. Middle: Elasticity mapping. Right: Surface elasticity measurements given by the Young's moduli shown in a histogram. (B) Comparison of 1,024 single surface elasticity measurements from individual MSCs shown in a box plot diagram ( n = 6).

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Examination of elasticity. (A) Left: Topographic images of membrane structures from MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL from passage 1 and 3 determined by atomic force microscopy. Middle: Elasticity mapping. Right: Surface elasticity measurements given by the Young's moduli shown in a histogram. (B) Comparison of 1,024 single surface elasticity measurements from individual MSCs shown in a box plot diagram ( n = 6).

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: Cell Culture, Microscopy

    Mitochondrial pattern analysis and super-resolution imaging. (A) Images of Mitochondrial Network Analysis (MiNA) from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with MitoTracker™ (B) Top: Numbers of punctate mitochondrial organelles per cell compared in different media and from passage 1 and 3, Wilcoxon and Mann Whitney test. Middle: Numbers of mitochondrial rods per cell, Mann Whitney test. Bottom: Numbers of mitochondrial networks per cell, Wilcoxon test. MiNA image interpretations are shown next to the diagrams. (C) Left: Median branch length of the mitochondrial networks in μm per cell, paired and unpaired t -test. Right: Mitochondrial footprint in μm 2 per cell. The same 30 cells per media or passage were compared in (B,C) . (D) Reconstructed fluorescence microscopy images (dSTORM) of mitochondria from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL. (E) Numbers of punctate mitochondrial organelles as well as rods and networks per cell from passage 3 in different media. Data were calculated with Mitochondrial Network Analysis (MiNA), image interpretations are shown next to the diagrams. * p

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Mitochondrial pattern analysis and super-resolution imaging. (A) Images of Mitochondrial Network Analysis (MiNA) from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with MitoTracker™ (B) Top: Numbers of punctate mitochondrial organelles per cell compared in different media and from passage 1 and 3, Wilcoxon and Mann Whitney test. Middle: Numbers of mitochondrial rods per cell, Mann Whitney test. Bottom: Numbers of mitochondrial networks per cell, Wilcoxon test. MiNA image interpretations are shown next to the diagrams. (C) Left: Median branch length of the mitochondrial networks in μm per cell, paired and unpaired t -test. Right: Mitochondrial footprint in μm 2 per cell. The same 30 cells per media or passage were compared in (B,C) . (D) Reconstructed fluorescence microscopy images (dSTORM) of mitochondria from passage 3 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL. (E) Numbers of punctate mitochondrial organelles as well as rods and networks per cell from passage 3 in different media. Data were calculated with Mitochondrial Network Analysis (MiNA), image interpretations are shown next to the diagrams. * p

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: Imaging, Cell Culture, Staining, MANN-WHITNEY, Fluorescence, Microscopy

    Expression of the immunological molecule GARP. (A) Relative gene expression levels of GARP after 24 and 48 h cultivation with MSCGM™ or MSCBM™ with 8% HPL and a control and a control at the beginning of cultivation time. Left: Normalized to the housekeeping gene HPRT (2D n = 5; 3D n = 3). Right: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) RT-PCR analysis of GARP gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (C) Left: GARP measurements of living CD90 + MSCs in 2D vs. 3D cultured in different media determined by flow cytometry, 6,000 cells were compared. Right: Dot plot of the mean fluorescence intensity of three batches in 2D and four batches in 3D, 6,000 MSCs were investigated per batch. (D) Box plot of the mean fluorescence intensity of GARP protein expression of five batches unstimulated or stimulated with IFN-γ/TNF-α for 24 and 48 h.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Expression of the immunological molecule GARP. (A) Relative gene expression levels of GARP after 24 and 48 h cultivation with MSCGM™ or MSCBM™ with 8% HPL and a control and a control at the beginning of cultivation time. Left: Normalized to the housekeeping gene HPRT (2D n = 5; 3D n = 3). Right: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (B) RT-PCR analysis of GARP gene expression in 2D and 3D MSCs for 24 and 48 h stimulation with IFN-γ/TNF-α cultured in MSCGM™ or MSCBM™ with 8% HPL and a control with unstimulated cells. Top: Normalized to the housekeeping gene HPRT. Bottom: Normalized to the housekeeping gene PPIA (2D n = 5; 3D n = 3). (C) Left: GARP measurements of living CD90 + MSCs in 2D vs. 3D cultured in different media determined by flow cytometry, 6,000 cells were compared. Right: Dot plot of the mean fluorescence intensity of three batches in 2D and four batches in 3D, 6,000 MSCs were investigated per batch. (D) Box plot of the mean fluorescence intensity of GARP protein expression of five batches unstimulated or stimulated with IFN-γ/TNF-α for 24 and 48 h.

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    Effect of HPL on 2D and 3D cultures. (A) Actin measurements with phalloidin of living CD90 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of four different batches for 3D MSCs and five batches for 2D MSCs of 4,000 cells per batch, one representative flow cytometry histogram is shown. (B) Mitochondria measurements with MitoTracker™ of living CD73 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of five different batches, 2,000 MSCs were investigated per batch, one representative flow cytometry histogram is shown. (C) Images of passage 1 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with the specific focal adhesion proteins vinculin and paxillin in green as well as stress fibers (f-actin) in red and nuclei in blue. Mitochondria were stained with MitoTracker™ in red, stress fibers in green and nuclei in blue. Type-3 intermediate filaments are given by staining vimentin in green, stress fibers in red and nuclei in blue. (D) Images of MSC spheroids, the staining corresponds to those described in (A) . The entire MSC spheroid is a maximal intensity projection of a stitched z-stack 3-channel overlay, magnification was 63x.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures

    doi: 10.3389/fbioe.2019.00338

    Figure Lengend Snippet: Effect of HPL on 2D and 3D cultures. (A) Actin measurements with phalloidin of living CD90 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of four different batches for 3D MSCs and five batches for 2D MSCs of 4,000 cells per batch, one representative flow cytometry histogram is shown. (B) Mitochondria measurements with MitoTracker™ of living CD73 + MSCs in 2D vs. 3D cultured in MSCGM™ or MSCBM™ with 8% HPL determined by flow cytometry. Dot plot of the mean fluorescence intensity of five different batches, 2,000 MSCs were investigated per batch, one representative flow cytometry histogram is shown. (C) Images of passage 1 MSCs cultured in MSCGM™ or MSCBM™ with 8% HPL stained with the specific focal adhesion proteins vinculin and paxillin in green as well as stress fibers (f-actin) in red and nuclei in blue. Mitochondria were stained with MitoTracker™ in red, stress fibers in green and nuclei in blue. Type-3 intermediate filaments are given by staining vimentin in green, stress fibers in red and nuclei in blue. (D) Images of MSC spheroids, the staining corresponds to those described in (A) . The entire MSC spheroid is a maximal intensity projection of a stitched z-stack 3-channel overlay, magnification was 63x.

    Article Snippet: Amnion derived MSCs propagated in adherence to plastic surface and analyzed in passage (P)1 or MSCs from spherical aggregates were positive for MSC specific markers CD73, CD90, and CD105 when cultivated in MSCGM™- medium or MSCBM™ with 8% HPL, and showed a size of ~30–60 μm in diameter ( ) compared to silica beads with similar refraction index.

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Fluorescence, Staining

    Human MSC grown in Plate-1 ( a, d ), Plate-2 ( b, e ), and controls ( b, f ) respectively (from left to right) imaged after 7 days of culture. In the top row shown are cells cultured using EC medium and in the bottom row are cells cultured using HMSC medium. Cells were stained using antibody for CD31 (green) and imaged using both phasecontrast and fluorescence. Scale bar in all images depicts 100 μm (color figure online)

    Journal: Cell biochemistry and biophysics

    Article Title: A Contact-Based Method for Differentiation of Human Mesenchymal Stem Cells into an Endothelial Cell-Phenotype

    doi: 10.1007/s12013-017-0828-z

    Figure Lengend Snippet: Human MSC grown in Plate-1 ( a, d ), Plate-2 ( b, e ), and controls ( b, f ) respectively (from left to right) imaged after 7 days of culture. In the top row shown are cells cultured using EC medium and in the bottom row are cells cultured using HMSC medium. Cells were stained using antibody for CD31 (green) and imaged using both phasecontrast and fluorescence. Scale bar in all images depicts 100 μm (color figure online)

    Article Snippet: To detect differentiation of human MSC into an EC-type, immunofluorescence detection for CD31 (or PECAM-1), an integral membrane glycoprotein that is expressed at high levels on endothelial cells, was performed [ ].

    Techniques: Cell Culture, Staining, Fluorescence

    Human MSC grown in Plate-1 ( a, d ), Plate-2 ( b, e ) and controls ( c, f ) respectively (from left to right) imaged after 7 days of culture. In the top row shown are cells cultured using EC medium and in the bottom row are cells cultured using HMSC medium. Cells were stained using antibody for CD31 (white) and imaged using both fluorescence. Scale bar in all images depicts 150 μm (color figure online)

    Journal: Cell biochemistry and biophysics

    Article Title: A Contact-Based Method for Differentiation of Human Mesenchymal Stem Cells into an Endothelial Cell-Phenotype

    doi: 10.1007/s12013-017-0828-z

    Figure Lengend Snippet: Human MSC grown in Plate-1 ( a, d ), Plate-2 ( b, e ) and controls ( c, f ) respectively (from left to right) imaged after 7 days of culture. In the top row shown are cells cultured using EC medium and in the bottom row are cells cultured using HMSC medium. Cells were stained using antibody for CD31 (white) and imaged using both fluorescence. Scale bar in all images depicts 150 μm (color figure online)

    Article Snippet: To detect differentiation of human MSC into an EC-type, immunofluorescence detection for CD31 (or PECAM-1), an integral membrane glycoprotein that is expressed at high levels on endothelial cells, was performed [ ].

    Techniques: Cell Culture, Staining, Fluorescence