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In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells.

Szaraz P, Librach M, Maghen L, Iqbal F, Barretto TA, Kenigsberg S, Gauthier-Fisher A, Librach CL - Stem Cells Int (2016)

Bottom Line: However the optimal cell type providing significant cardiac regeneration after MI is yet to be found.Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity.Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

View Article: PubMed Central - PubMed

Affiliation: Create Fertility Centre, Toronto, ON, Canada M5G 1N8; Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada M5S 1A8.

ABSTRACT
Myocardial infarction (MI) causes an extensive loss of heart muscle cells and leads to congestive heart disease (CAD), the leading cause of mortality and morbidity worldwide. Mesenchymal stromal cell- (MSC-) based cell therapy is a promising option to replace invasive interventions. However the optimal cell type providing significant cardiac regeneration after MI is yet to be found. The aim of our study was to investigate the cardiomyogenic differentiation potential of first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel, young source of immunoprivileged mesenchymal stromal cells. Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity. When applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells within 1 week of coculture, making them the first MSC type with this ability. Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

No MeSH data available.


Related in: MedlinePlus

In vitro cardiomyogenic differentiation of human MSCs. (a–d) FC analysis of hMSCs. Undifferentiated MSC markers SSEA4 (a) and CD146 (b), cardiomyocyte marker SIRPA (c), and gap junction protein connexin 43 (cx43, (d)) levels expressed as % of overall human cell counts in undifferentiated and differentiated (direct coculture, aggregate coculture) first trimester PVC (FTM), term HUCPVC (term), and bone marrow (BMSC). ∗ indicates statistically significant difference (p < 0.01, nFTM = 9, nterm = 9, nBMSC = 6). (e) Confocal microscopy images of Mef2c (green) and HuNu (red) immunostaining in FTM and term HUCPVCs and bone marrow MSC containing rat primary cardiomyocyte cultures in comparison with undifferentiated MSCs. Blue: Hoechst (DAPI) Arrows mark double positive nuclei. Bar = 100 μm. (f) Quantification of Mef2c and HuNu double positive nuclei in direct cocultures, expressed as % of HuNu positive (overall hMSC) counts. 50 independent, random fields of sight assessed per MSC type. ∗p < 0.01. (g–i) Confocal microscopy images of TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures compared with undifferentiated cultures. Connexin 43 ((h, i), red) and cardiac troponin T ((g, i), green) stainings. For connexin 43, cells were counterstained with fixable dye (CellTracker Green). (h) Inner box: 2x magnification of representative fields showing intracellular distribution of cx43 positive puncta. Scale bars (e, i): 100 μm; (g, h): x = 54 μm and y = 48 μm and x = 100 μm and y = 110 μm. Blue: Hoechst (DAPI filter). (j, k) Quantitative PCR analysis of cardiac troponin T (cTnT, (j)) and heavy chain cardiac myosin (MYH6, (k)) in TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures. Values expressed as fold change in comparison to undifferentiated cells. n = 4.
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fig2: In vitro cardiomyogenic differentiation of human MSCs. (a–d) FC analysis of hMSCs. Undifferentiated MSC markers SSEA4 (a) and CD146 (b), cardiomyocyte marker SIRPA (c), and gap junction protein connexin 43 (cx43, (d)) levels expressed as % of overall human cell counts in undifferentiated and differentiated (direct coculture, aggregate coculture) first trimester PVC (FTM), term HUCPVC (term), and bone marrow (BMSC). ∗ indicates statistically significant difference (p < 0.01, nFTM = 9, nterm = 9, nBMSC = 6). (e) Confocal microscopy images of Mef2c (green) and HuNu (red) immunostaining in FTM and term HUCPVCs and bone marrow MSC containing rat primary cardiomyocyte cultures in comparison with undifferentiated MSCs. Blue: Hoechst (DAPI) Arrows mark double positive nuclei. Bar = 100 μm. (f) Quantification of Mef2c and HuNu double positive nuclei in direct cocultures, expressed as % of HuNu positive (overall hMSC) counts. 50 independent, random fields of sight assessed per MSC type. ∗p < 0.01. (g–i) Confocal microscopy images of TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures compared with undifferentiated cultures. Connexin 43 ((h, i), red) and cardiac troponin T ((g, i), green) stainings. For connexin 43, cells were counterstained with fixable dye (CellTracker Green). (h) Inner box: 2x magnification of representative fields showing intracellular distribution of cx43 positive puncta. Scale bars (e, i): 100 μm; (g, h): x = 54 μm and y = 48 μm and x = 100 μm and y = 110 μm. Blue: Hoechst (DAPI filter). (j, k) Quantitative PCR analysis of cardiac troponin T (cTnT, (j)) and heavy chain cardiac myosin (MYH6, (k)) in TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures. Values expressed as fold change in comparison to undifferentiated cells. n = 4.

Mentions: We analysed TRA-1-85high cells for the expression of multipotent stromal cell marker SSEA4 [55], pericyte, and adult stem cell marker CD146 [56], as well as cardiomyocyte markers SIRPA and CX43 using FC. The majority of TRA-1-85high MSCs (FTM, term, and BMSC) significantly downregulated SSEA4 expression in both direct coculture and aggregate coculture when compared to undifferentiated cells (Figure 2(a), p < 0.01). The proportion of CD146-positive cells significantly decreased during both direct coculture and aggregate coculture differentiations (Figure 2(b), p < 0.01). All 3 undifferentiated MSC types were found to be ≥90% CD146-positive and this significantly (p < 0.01) decreased in direct cocultures (26.9% ± 9.3%, 29.6% ± 15%, 27.6% ± 14%, resp.). The percentage of CD146 +ve cells further decreased in term HUCPVC and BMSC aggregate cocultures (13.6% ± 8% and 19.5% ± 8.2%, resp.), but not in FTM HUCPVC cocultures (30.4% ± 16.9%) (Figure 2(b)). Cardiomyocyte-associated marker SIRPA was upregulated in FTM HUCPVCs (35.6% ± 6.9%) and term HUCPVCs (57.9% ± 14.7%), but not in BMSCs (5.3% ± 2.6%) (Figure 2(c)). Significantly more FTM (35% ± 13%) compared to term (19.2% ± 7.8%) and BMSC (13.2% ± 5.8%) upregulated connexin 43 (cx43) (p < 0.01) (Figure 2(c)). Aggregate cocultures further increased SIRPA levels in term HUCPVCs (57.9% ± 14.7%) to significantly higher levels (p < 0.01) when compared to FTM (35.6% ± 6.9%) and BMSC (17.6% ± 3.4%). Concurrently, cx43 positivity of term HUCPVCs became significantly higher (72.3% ± 2.6%) than either FTM (37.2% ± 13%) or BMSC (3.8% ± 1.9%) in this assay (Figure 2(d)).


In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells.

Szaraz P, Librach M, Maghen L, Iqbal F, Barretto TA, Kenigsberg S, Gauthier-Fisher A, Librach CL - Stem Cells Int (2016)

In vitro cardiomyogenic differentiation of human MSCs. (a–d) FC analysis of hMSCs. Undifferentiated MSC markers SSEA4 (a) and CD146 (b), cardiomyocyte marker SIRPA (c), and gap junction protein connexin 43 (cx43, (d)) levels expressed as % of overall human cell counts in undifferentiated and differentiated (direct coculture, aggregate coculture) first trimester PVC (FTM), term HUCPVC (term), and bone marrow (BMSC). ∗ indicates statistically significant difference (p < 0.01, nFTM = 9, nterm = 9, nBMSC = 6). (e) Confocal microscopy images of Mef2c (green) and HuNu (red) immunostaining in FTM and term HUCPVCs and bone marrow MSC containing rat primary cardiomyocyte cultures in comparison with undifferentiated MSCs. Blue: Hoechst (DAPI) Arrows mark double positive nuclei. Bar = 100 μm. (f) Quantification of Mef2c and HuNu double positive nuclei in direct cocultures, expressed as % of HuNu positive (overall hMSC) counts. 50 independent, random fields of sight assessed per MSC type. ∗p < 0.01. (g–i) Confocal microscopy images of TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures compared with undifferentiated cultures. Connexin 43 ((h, i), red) and cardiac troponin T ((g, i), green) stainings. For connexin 43, cells were counterstained with fixable dye (CellTracker Green). (h) Inner box: 2x magnification of representative fields showing intracellular distribution of cx43 positive puncta. Scale bars (e, i): 100 μm; (g, h): x = 54 μm and y = 48 μm and x = 100 μm and y = 110 μm. Blue: Hoechst (DAPI filter). (j, k) Quantitative PCR analysis of cardiac troponin T (cTnT, (j)) and heavy chain cardiac myosin (MYH6, (k)) in TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures. Values expressed as fold change in comparison to undifferentiated cells. n = 4.
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Related In: Results  -  Collection

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fig2: In vitro cardiomyogenic differentiation of human MSCs. (a–d) FC analysis of hMSCs. Undifferentiated MSC markers SSEA4 (a) and CD146 (b), cardiomyocyte marker SIRPA (c), and gap junction protein connexin 43 (cx43, (d)) levels expressed as % of overall human cell counts in undifferentiated and differentiated (direct coculture, aggregate coculture) first trimester PVC (FTM), term HUCPVC (term), and bone marrow (BMSC). ∗ indicates statistically significant difference (p < 0.01, nFTM = 9, nterm = 9, nBMSC = 6). (e) Confocal microscopy images of Mef2c (green) and HuNu (red) immunostaining in FTM and term HUCPVCs and bone marrow MSC containing rat primary cardiomyocyte cultures in comparison with undifferentiated MSCs. Blue: Hoechst (DAPI) Arrows mark double positive nuclei. Bar = 100 μm. (f) Quantification of Mef2c and HuNu double positive nuclei in direct cocultures, expressed as % of HuNu positive (overall hMSC) counts. 50 independent, random fields of sight assessed per MSC type. ∗p < 0.01. (g–i) Confocal microscopy images of TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures compared with undifferentiated cultures. Connexin 43 ((h, i), red) and cardiac troponin T ((g, i), green) stainings. For connexin 43, cells were counterstained with fixable dye (CellTracker Green). (h) Inner box: 2x magnification of representative fields showing intracellular distribution of cx43 positive puncta. Scale bars (e, i): 100 μm; (g, h): x = 54 μm and y = 48 μm and x = 100 μm and y = 110 μm. Blue: Hoechst (DAPI filter). (j, k) Quantitative PCR analysis of cardiac troponin T (cTnT, (j)) and heavy chain cardiac myosin (MYH6, (k)) in TRA-1-85high FTM and term HUCPVCs sorted from direct cocultures. Values expressed as fold change in comparison to undifferentiated cells. n = 4.
Mentions: We analysed TRA-1-85high cells for the expression of multipotent stromal cell marker SSEA4 [55], pericyte, and adult stem cell marker CD146 [56], as well as cardiomyocyte markers SIRPA and CX43 using FC. The majority of TRA-1-85high MSCs (FTM, term, and BMSC) significantly downregulated SSEA4 expression in both direct coculture and aggregate coculture when compared to undifferentiated cells (Figure 2(a), p < 0.01). The proportion of CD146-positive cells significantly decreased during both direct coculture and aggregate coculture differentiations (Figure 2(b), p < 0.01). All 3 undifferentiated MSC types were found to be ≥90% CD146-positive and this significantly (p < 0.01) decreased in direct cocultures (26.9% ± 9.3%, 29.6% ± 15%, 27.6% ± 14%, resp.). The percentage of CD146 +ve cells further decreased in term HUCPVC and BMSC aggregate cocultures (13.6% ± 8% and 19.5% ± 8.2%, resp.), but not in FTM HUCPVC cocultures (30.4% ± 16.9%) (Figure 2(b)). Cardiomyocyte-associated marker SIRPA was upregulated in FTM HUCPVCs (35.6% ± 6.9%) and term HUCPVCs (57.9% ± 14.7%), but not in BMSCs (5.3% ± 2.6%) (Figure 2(c)). Significantly more FTM (35% ± 13%) compared to term (19.2% ± 7.8%) and BMSC (13.2% ± 5.8%) upregulated connexin 43 (cx43) (p < 0.01) (Figure 2(c)). Aggregate cocultures further increased SIRPA levels in term HUCPVCs (57.9% ± 14.7%) to significantly higher levels (p < 0.01) when compared to FTM (35.6% ± 6.9%) and BMSC (17.6% ± 3.4%). Concurrently, cx43 positivity of term HUCPVCs became significantly higher (72.3% ± 2.6%) than either FTM (37.2% ± 13%) or BMSC (3.8% ± 1.9%) in this assay (Figure 2(d)).

Bottom Line: However the optimal cell type providing significant cardiac regeneration after MI is yet to be found.Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity.Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

View Article: PubMed Central - PubMed

Affiliation: Create Fertility Centre, Toronto, ON, Canada M5G 1N8; Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada M5S 1A8.

ABSTRACT
Myocardial infarction (MI) causes an extensive loss of heart muscle cells and leads to congestive heart disease (CAD), the leading cause of mortality and morbidity worldwide. Mesenchymal stromal cell- (MSC-) based cell therapy is a promising option to replace invasive interventions. However the optimal cell type providing significant cardiac regeneration after MI is yet to be found. The aim of our study was to investigate the cardiomyogenic differentiation potential of first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel, young source of immunoprivileged mesenchymal stromal cells. Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity. When applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells within 1 week of coculture, making them the first MSC type with this ability. Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

No MeSH data available.


Related in: MedlinePlus