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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

Substrate signaling from 70:30 FN:LN compared with gelatin increased the number of p-FAK, p-ERK, ITGB4, and ITGB5 positive cells. Percentages of ITGB 4, ITGB 5, p-FAK, and p-ERK expressing cells from (A) H7and (B) H9 hESCs differentiating on 70:30 FN:LN were higher than those of cells on gelatin at day 14. Bars indicate statistically significant differences between the indicated groups. p-FAK, phosphorylated focal adhesion kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; ITGB, intergrin β.
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f3: Substrate signaling from 70:30 FN:LN compared with gelatin increased the number of p-FAK, p-ERK, ITGB4, and ITGB5 positive cells. Percentages of ITGB 4, ITGB 5, p-FAK, and p-ERK expressing cells from (A) H7and (B) H9 hESCs differentiating on 70:30 FN:LN were higher than those of cells on gelatin at day 14. Bars indicate statistically significant differences between the indicated groups. p-FAK, phosphorylated focal adhesion kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; ITGB, intergrin β.

Mentions: The ITGB4 and ITGB5 protein expression in H7 and H9 differentiation cultures on the optimal substrate and gelatin were first analyzed by flow cytometry analysis. The H7 differentiated on the optimal substrate, 70:30 FN:LN, contained 21% ITGB4 and 31% ITGB5 on day 14; whereas, the same cells on the gelatin contained 6% ITGB4 land 10% ITGB5 (Fig. 3A, Supplementary Fig. S2A). Similarly, H9 ESC differentiating cells on the FN/LN substrate contained higher numbers, 27% of ITGB4+ cells and 44% ITGB5+ cells versus 12% and 13%, respectively, when derived on gelatin (Fig. 3B, Supplementary Fig. S2B). Evidence of downstream integrin signaling was also investigated by analyzing the phosphorylation of both FAK and ERK in cells differentiated on the 70:30 FN:LN compared with gelatin. The significantly increased numbers of p-FAK (25% vs. 9% in H7; 27% vs. 12% in H9) and p-ERK (23% vs. 15% in H7; 31% vs. 8% in H9) were detected in hESCs differentiated on the optimal substrate compared with gelatin, respectively (Fig. 3, Supplementary Fig. S2).


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)

Substrate signaling from 70:30 FN:LN compared with gelatin increased the number of p-FAK, p-ERK, ITGB4, and ITGB5 positive cells. Percentages of ITGB 4, ITGB 5, p-FAK, and p-ERK expressing cells from (A) H7and (B) H9 hESCs differentiating on 70:30 FN:LN were higher than those of cells on gelatin at day 14. Bars indicate statistically significant differences between the indicated groups. p-FAK, phosphorylated focal adhesion kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; ITGB, intergrin β.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4120929&req=5

f3: Substrate signaling from 70:30 FN:LN compared with gelatin increased the number of p-FAK, p-ERK, ITGB4, and ITGB5 positive cells. Percentages of ITGB 4, ITGB 5, p-FAK, and p-ERK expressing cells from (A) H7and (B) H9 hESCs differentiating on 70:30 FN:LN were higher than those of cells on gelatin at day 14. Bars indicate statistically significant differences between the indicated groups. p-FAK, phosphorylated focal adhesion kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; ITGB, intergrin β.
Mentions: The ITGB4 and ITGB5 protein expression in H7 and H9 differentiation cultures on the optimal substrate and gelatin were first analyzed by flow cytometry analysis. The H7 differentiated on the optimal substrate, 70:30 FN:LN, contained 21% ITGB4 and 31% ITGB5 on day 14; whereas, the same cells on the gelatin contained 6% ITGB4 land 10% ITGB5 (Fig. 3A, Supplementary Fig. S2A). Similarly, H9 ESC differentiating cells on the FN/LN substrate contained higher numbers, 27% of ITGB4+ cells and 44% ITGB5+ cells versus 12% and 13%, respectively, when derived on gelatin (Fig. 3B, Supplementary Fig. S2B). Evidence of downstream integrin signaling was also investigated by analyzing the phosphorylation of both FAK and ERK in cells differentiated on the 70:30 FN:LN compared with gelatin. The significantly increased numbers of p-FAK (25% vs. 9% in H7; 27% vs. 12% in H9) and p-ERK (23% vs. 15% in H7; 31% vs. 8% in H9) were detected in hESCs differentiated on the optimal substrate compared with gelatin, respectively (Fig. 3, Supplementary Fig. S2).

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