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Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells.

Sa S, Wong L, McCloskey KE - Biores Open Access (2014)

Bottom Line: Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin.Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway.Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate Group in Biological Engineering & Small-Scale Technologies, University of California , Merced, California.

ABSTRACT
Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.

No MeSH data available.


Related in: MedlinePlus

Blocking ITGB4 and/or ITGB5 reduced both the percentage of cardiomyocyte (CM) positive cells and p-FAK–expressing cells in differentiating hESC cultures. The reduced numbers of cTnI+ and Nkx2.5+ cells in differentiated hESC cultures (A) H7 and (B) H9 treated with ITGB4 and/or ITGB5 blocking antibodies at day 14 correlate with the reduction in p-FAK. Blocking MEK signaling inhibited cardiomyocyte differentiation from H7 (C) and H9 (D). The cTnI+, Nkx2.5+, and p-ERK+ cells in differentiation cultures were significantly much greater compared with cultures treated with MEK inhibitor U0126 at day 14. Bars indicate statistically significant differences between the indicated groups.
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f4: Blocking ITGB4 and/or ITGB5 reduced both the percentage of cardiomyocyte (CM) positive cells and p-FAK–expressing cells in differentiating hESC cultures. The reduced numbers of cTnI+ and Nkx2.5+ cells in differentiated hESC cultures (A) H7 and (B) H9 treated with ITGB4 and/or ITGB5 blocking antibodies at day 14 correlate with the reduction in p-FAK. Blocking MEK signaling inhibited cardiomyocyte differentiation from H7 (C) and H9 (D). The cTnI+, Nkx2.5+, and p-ERK+ cells in differentiation cultures were significantly much greater compared with cultures treated with MEK inhibitor U0126 at day 14. Bars indicate statistically significant differences between the indicated groups.

Mentions: In order to verify the role of ITGB4 and ITGB5 signaling in CM differentiation, cells were treated with ITGB4 and/or ITGB5 blocking antibodies starting from the day the cells were plated as a monolayer on the 70:30 FN:LN (optimized at day 5 and 6 for H7 and H9, respectively16) through day 14. ITGB4 blocking antibody treatment reduced cTnI+ cells in H7 and H9 by 50% and 45%, respectively, and reduced Nkx2.5+ cells in H7 and H9 by 74% and 64%, respectively (Fig. 4, Supplementary Fig. S3). ITGB5 blocking antibody treatment reduced cTnI and Nkx2.5 expression in H7 and H9 by even more, 83%–89% (Fig. 4, Supplementary Fig. S3). Blocking both ITGB4 and ITGB5 simultaneously almost completely eliminated the CM derivation in these culture, reducing cTnI and Nkx2.5 expression in both H7 and H9 by 92%–97% (Fig. 4, Supplementary Fig. S3).


Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells.

Sa S, Wong L, McCloskey KE - Biores Open Access (2014)

Blocking ITGB4 and/or ITGB5 reduced both the percentage of cardiomyocyte (CM) positive cells and p-FAK–expressing cells in differentiating hESC cultures. The reduced numbers of cTnI+ and Nkx2.5+ cells in differentiated hESC cultures (A) H7 and (B) H9 treated with ITGB4 and/or ITGB5 blocking antibodies at day 14 correlate with the reduction in p-FAK. Blocking MEK signaling inhibited cardiomyocyte differentiation from H7 (C) and H9 (D). The cTnI+, Nkx2.5+, and p-ERK+ cells in differentiation cultures were significantly much greater compared with cultures treated with MEK inhibitor U0126 at day 14. Bars indicate statistically significant differences between the indicated groups.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4120929&req=5

f4: Blocking ITGB4 and/or ITGB5 reduced both the percentage of cardiomyocyte (CM) positive cells and p-FAK–expressing cells in differentiating hESC cultures. The reduced numbers of cTnI+ and Nkx2.5+ cells in differentiated hESC cultures (A) H7 and (B) H9 treated with ITGB4 and/or ITGB5 blocking antibodies at day 14 correlate with the reduction in p-FAK. Blocking MEK signaling inhibited cardiomyocyte differentiation from H7 (C) and H9 (D). The cTnI+, Nkx2.5+, and p-ERK+ cells in differentiation cultures were significantly much greater compared with cultures treated with MEK inhibitor U0126 at day 14. Bars indicate statistically significant differences between the indicated groups.
Mentions: In order to verify the role of ITGB4 and ITGB5 signaling in CM differentiation, cells were treated with ITGB4 and/or ITGB5 blocking antibodies starting from the day the cells were plated as a monolayer on the 70:30 FN:LN (optimized at day 5 and 6 for H7 and H9, respectively16) through day 14. ITGB4 blocking antibody treatment reduced cTnI+ cells in H7 and H9 by 50% and 45%, respectively, and reduced Nkx2.5+ cells in H7 and H9 by 74% and 64%, respectively (Fig. 4, Supplementary Fig. S3). ITGB5 blocking antibody treatment reduced cTnI and Nkx2.5 expression in H7 and H9 by even more, 83%–89% (Fig. 4, Supplementary Fig. S3). Blocking both ITGB4 and ITGB5 simultaneously almost completely eliminated the CM derivation in these culture, reducing cTnI and Nkx2.5 expression in both H7 and H9 by 92%–97% (Fig. 4, Supplementary Fig. S3).

Bottom Line: Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin.Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway.Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate Group in Biological Engineering & Small-Scale Technologies, University of California , Merced, California.

ABSTRACT
Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.

No MeSH data available.


Related in: MedlinePlus