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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

Immunofluorescent images of hESC-CM. The (A) H7 and (B) H9 ESC-derived CMs were imaged under confocal microscope at day 14. The cells were immunofluorescently stained with cardiac troponin I (green) and DAPI (blue). The presence of some sacromeric structures are indicated by arrows. (C) The differentiated CM cultures from H9 ESCs were also co-stained with cTnI TRITC (red), ITGB5 FITC (green), and DAPI (blue), as well as (D) co-stained with ITGB5 FITC (green), ITGB4 (red), and DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.
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f5: Immunofluorescent images of hESC-CM. The (A) H7 and (B) H9 ESC-derived CMs were imaged under confocal microscope at day 14. The cells were immunofluorescently stained with cardiac troponin I (green) and DAPI (blue). The presence of some sacromeric structures are indicated by arrows. (C) The differentiated CM cultures from H9 ESCs were also co-stained with cTnI TRITC (red), ITGB5 FITC (green), and DAPI (blue), as well as (D) co-stained with ITGB5 FITC (green), ITGB4 (red), and DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.

Mentions: The differentiated cultures were stained for cTnI and imaged in order to verify the presence of sacromeric structures (Fig. 5A, 5B). The differentiated CM cultures were then co-stained with cTnI tetramethyl rhodamine isothiocyanate (TRITC), ITGB5 fluorescein isothiocyanate (FITC), and DAPI (Fig. 5C). The image shows that cTnI+ cells were also positive for ITGB5 FITC. Interestingly, the co-staining of ITGB5 FITC with ITGB4 TRITC indicates that the cells are not double-positive for both integrins, but that the CM cultures contain cells expressing either ITGB4 or ITGB5 (Fig. 5D). Consistent with FACS measurements (Fig. 3), this image depicts that the cultures contain greater numbers of cells expressing ITGB5 compared with ITGB4, and that the spatial patterning of the ITGB4 and ITGB5 cells is mingled such that each ITGB5 cells is in contact with the ITGB4-expressing cells.


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)

Immunofluorescent images of hESC-CM. The (A) H7 and (B) H9 ESC-derived CMs were imaged under confocal microscope at day 14. The cells were immunofluorescently stained with cardiac troponin I (green) and DAPI (blue). The presence of some sacromeric structures are indicated by arrows. (C) The differentiated CM cultures from H9 ESCs were also co-stained with cTnI TRITC (red), ITGB5 FITC (green), and DAPI (blue), as well as (D) co-stained with ITGB5 FITC (green), ITGB4 (red), and DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4120929&req=5

f5: Immunofluorescent images of hESC-CM. The (A) H7 and (B) H9 ESC-derived CMs were imaged under confocal microscope at day 14. The cells were immunofluorescently stained with cardiac troponin I (green) and DAPI (blue). The presence of some sacromeric structures are indicated by arrows. (C) The differentiated CM cultures from H9 ESCs were also co-stained with cTnI TRITC (red), ITGB5 FITC (green), and DAPI (blue), as well as (D) co-stained with ITGB5 FITC (green), ITGB4 (red), and DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.
Mentions: The differentiated cultures were stained for cTnI and imaged in order to verify the presence of sacromeric structures (Fig. 5A, 5B). The differentiated CM cultures were then co-stained with cTnI tetramethyl rhodamine isothiocyanate (TRITC), ITGB5 fluorescein isothiocyanate (FITC), and DAPI (Fig. 5C). The image shows that cTnI+ cells were also positive for ITGB5 FITC. Interestingly, the co-staining of ITGB5 FITC with ITGB4 TRITC indicates that the cells are not double-positive for both integrins, but that the CM cultures contain cells expressing either ITGB4 or ITGB5 (Fig. 5D). Consistent with FACS measurements (Fig. 3), this image depicts that the cultures contain greater numbers of cells expressing ITGB5 compared with ITGB4, and that the spatial patterning of the ITGB4 and ITGB5 cells is mingled such that each ITGB5 cells is in contact with the ITGB4-expressing cells.

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