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Innate immunity in human embryonic stem cells: comparison with adult human endothelial cells.

Földes G, Liu A, Badiger R, Paul-Clark M, Moreno L, Lendvai Z, Wright JS, Ali NN, Harding SE, Mitchell JA - PLoS ONE (2010)

Bottom Line: Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals.TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin.These findings have potential implications for survival and function of grafted hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: National Heart and Lung Institute, Imperial College, London, United Kingdom.

ABSTRACT
Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.

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Presence of cardiomyocytes and endothelial cells in differentiated hESC cultures.Immunocytochemical staining of clusters of 1 month differentiated H7 hESC showing (a) cardiomyocytes myosin heavy chain (α, β) (green) with corresponding brightfield image and (b) endothelial cells identified with von Willebrand factor (green), CD31 (red) and DAPI (nuclei, blue).
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pone-0010501-g002: Presence of cardiomyocytes and endothelial cells in differentiated hESC cultures.Immunocytochemical staining of clusters of 1 month differentiated H7 hESC showing (a) cardiomyocytes myosin heavy chain (α, β) (green) with corresponding brightfield image and (b) endothelial cells identified with von Willebrand factor (green), CD31 (red) and DAPI (nuclei, blue).

Mentions: Undifferentiated hESC have a characteristic appearance as tightly packed cells in colonies as shown in Fig. 1a and b. The H7 line, obtained from Geron Corp., Menlo Park CA, was grown under feeder-free conditions as described previously [15] and differentiated via embryoid body formation in 20% FCS. After four days of differentiation in suspension cultures, embryoid bodies were plated out onto gelatinized surfaces and continued to differentiate in adherent cultures for prolonged periods (over 4 months). Figure 1C-F shows the morphology of cultures at 1 and 3 months after differentiation, demonstrating the emergence of a variety of features, including clusters of beating cardiomyocytes (video S1) and vessel-like structures. Immunocytochemical staining for known markers (Fig. 2) confirms the presence of cardiomyocytes and endothelial cells within the mixed population of cells.


Innate immunity in human embryonic stem cells: comparison with adult human endothelial cells.

Földes G, Liu A, Badiger R, Paul-Clark M, Moreno L, Lendvai Z, Wright JS, Ali NN, Harding SE, Mitchell JA - PLoS ONE (2010)

Presence of cardiomyocytes and endothelial cells in differentiated hESC cultures.Immunocytochemical staining of clusters of 1 month differentiated H7 hESC showing (a) cardiomyocytes myosin heavy chain (α, β) (green) with corresponding brightfield image and (b) endothelial cells identified with von Willebrand factor (green), CD31 (red) and DAPI (nuclei, blue).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864770&req=5

pone-0010501-g002: Presence of cardiomyocytes and endothelial cells in differentiated hESC cultures.Immunocytochemical staining of clusters of 1 month differentiated H7 hESC showing (a) cardiomyocytes myosin heavy chain (α, β) (green) with corresponding brightfield image and (b) endothelial cells identified with von Willebrand factor (green), CD31 (red) and DAPI (nuclei, blue).
Mentions: Undifferentiated hESC have a characteristic appearance as tightly packed cells in colonies as shown in Fig. 1a and b. The H7 line, obtained from Geron Corp., Menlo Park CA, was grown under feeder-free conditions as described previously [15] and differentiated via embryoid body formation in 20% FCS. After four days of differentiation in suspension cultures, embryoid bodies were plated out onto gelatinized surfaces and continued to differentiate in adherent cultures for prolonged periods (over 4 months). Figure 1C-F shows the morphology of cultures at 1 and 3 months after differentiation, demonstrating the emergence of a variety of features, including clusters of beating cardiomyocytes (video S1) and vessel-like structures. Immunocytochemical staining for known markers (Fig. 2) confirms the presence of cardiomyocytes and endothelial cells within the mixed population of cells.

Bottom Line: Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals.TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin.These findings have potential implications for survival and function of grafted hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: National Heart and Lung Institute, Imperial College, London, United Kingdom.

ABSTRACT
Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.

Show MeSH