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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

Human extracellular matrix (ECM) and adhesion molecules gene expression of cells cultured on 70:30 FN:LN compared with gelatin. Volcano plots H7 (A) and H9 (B) compared ECM and adhesion molecules gene expression fold change between cells cultured on optimal substrate and cells cultured on gelatin. The x-axis indicates the fold change, while the y-axis indicates the p-value of the fold change. Each dot represents the expression of one gene. Dots above the horizontal line show significant difference (p<0.1). Dots in red represent up-regulated genes, dots in green represent down-regulated genes, while dots in black represent unchanged genes.
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f2: Human extracellular matrix (ECM) and adhesion molecules gene expression of cells cultured on 70:30 FN:LN compared with gelatin. Volcano plots H7 (A) and H9 (B) compared ECM and adhesion molecules gene expression fold change between cells cultured on optimal substrate and cells cultured on gelatin. The x-axis indicates the fold change, while the y-axis indicates the p-value of the fold change. Each dot represents the expression of one gene. Dots above the horizontal line show significant difference (p<0.1). Dots in red represent up-regulated genes, dots in green represent down-regulated genes, while dots in black represent unchanged genes.

Mentions: To further understand the molecules involved in FN/LN signaling, we evaluated the gene expression of using a quantitative reverse-transcription PCR micoarray that simultaneously analyzed the gene expression of 84 ECM and adhesion molecules. Here, we compared our optimal substrate 70:30 FN:LN against gelatin (control) based on the fact that some of the most efficient protocols culture cells on gelatin during CM differentiation.13 Based on volcano plots of the array data, 12 genes were significantly up-regulated (p<0.1) in H7, and 25 genes were significantly up-regulated in H9 (Fig. 2, Supplementary Table S1). Based on the categorization of the gene products; 5 out of 12 of the up-regulated genes in H7-derived CMs and 8 out of 25 of the up-regulated genes in H9-derived CMs were transmembrane molecules. The remaining up-regulated genes were adhesion molecules (three and four); ECM proteases (one and five); and cell–cell adhesion molecules (one and four) in H7 and H9, respectively. Based on these data, we deduced that the transmembrane molecules may play the most important roles for directing CM fate.


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)

Human extracellular matrix (ECM) and adhesion molecules gene expression of cells cultured on 70:30 FN:LN compared with gelatin. Volcano plots H7 (A) and H9 (B) compared ECM and adhesion molecules gene expression fold change between cells cultured on optimal substrate and cells cultured on gelatin. The x-axis indicates the fold change, while the y-axis indicates the p-value of the fold change. Each dot represents the expression of one gene. Dots above the horizontal line show significant difference (p<0.1). Dots in red represent up-regulated genes, dots in green represent down-regulated genes, while dots in black represent unchanged genes.
© Copyright Policy
Related In: Results  -  Collection

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

f2: Human extracellular matrix (ECM) and adhesion molecules gene expression of cells cultured on 70:30 FN:LN compared with gelatin. Volcano plots H7 (A) and H9 (B) compared ECM and adhesion molecules gene expression fold change between cells cultured on optimal substrate and cells cultured on gelatin. The x-axis indicates the fold change, while the y-axis indicates the p-value of the fold change. Each dot represents the expression of one gene. Dots above the horizontal line show significant difference (p<0.1). Dots in red represent up-regulated genes, dots in green represent down-regulated genes, while dots in black represent unchanged genes.
Mentions: To further understand the molecules involved in FN/LN signaling, we evaluated the gene expression of using a quantitative reverse-transcription PCR micoarray that simultaneously analyzed the gene expression of 84 ECM and adhesion molecules. Here, we compared our optimal substrate 70:30 FN:LN against gelatin (control) based on the fact that some of the most efficient protocols culture cells on gelatin during CM differentiation.13 Based on volcano plots of the array data, 12 genes were significantly up-regulated (p<0.1) in H7, and 25 genes were significantly up-regulated in H9 (Fig. 2, Supplementary Table S1). Based on the categorization of the gene products; 5 out of 12 of the up-regulated genes in H7-derived CMs and 8 out of 25 of the up-regulated genes in H9-derived CMs were transmembrane molecules. The remaining up-regulated genes were adhesion molecules (three and four); ECM proteases (one and five); and cell–cell adhesion molecules (one and four) in H7 and H9, respectively. Based on these data, we deduced that the transmembrane molecules may play the most important roles for directing CM fate.

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