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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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Expression of TLR genes in differentiated hESC.Relative expression in differentiated H7 hESC at 1 month (n = 2, open bar), 3 months (grey bar) and 4 months (solid bar) compared to A: averaged undifferentiated H7 hESC (n = 3) and B: human aortic endothelial cells (HAEC, n = 3).
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pone-0010501-g005: Expression of TLR genes in differentiated hESC.Relative expression in differentiated H7 hESC at 1 month (n = 2, open bar), 3 months (grey bar) and 4 months (solid bar) compared to A: averaged undifferentiated H7 hESC (n = 3) and B: human aortic endothelial cells (HAEC, n = 3).

Mentions: Expression levels were determined at 1, 3 and 4 months after differentiation of H7 hESC. Compared to undifferentiated H7 (Fig. 5A), there was a general increase with time in TLRs 1, 2, 3, 4, 5 and 6, with all above undifferentiated levels by 4 months. Low levels of TLRs 7, 9 and 10 also became sporadically apparent (Table S1). However, comparing differentiated hESC with endothelial cells (Fig. 5B) it is clear that TLR1 and TLR4 were still at low levels, even after 4 months of differentiation. Only modest adjustments of expression level of the NFκB (Table 1) levels were seen during differentiation while some components of the TLR signaling pathways (TICAM1, TICAM2 and IRAK1) were consistently increased (Table 2). A full list of gene expression changes during differentiation is found in Table S2.


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)

Expression of TLR genes in differentiated hESC.Relative expression in differentiated H7 hESC at 1 month (n = 2, open bar), 3 months (grey bar) and 4 months (solid bar) compared to A: averaged undifferentiated H7 hESC (n = 3) and B: human aortic endothelial cells (HAEC, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010501-g005: Expression of TLR genes in differentiated hESC.Relative expression in differentiated H7 hESC at 1 month (n = 2, open bar), 3 months (grey bar) and 4 months (solid bar) compared to A: averaged undifferentiated H7 hESC (n = 3) and B: human aortic endothelial cells (HAEC, n = 3).
Mentions: Expression levels were determined at 1, 3 and 4 months after differentiation of H7 hESC. Compared to undifferentiated H7 (Fig. 5A), there was a general increase with time in TLRs 1, 2, 3, 4, 5 and 6, with all above undifferentiated levels by 4 months. Low levels of TLRs 7, 9 and 10 also became sporadically apparent (Table S1). However, comparing differentiated hESC with endothelial cells (Fig. 5B) it is clear that TLR1 and TLR4 were still at low levels, even after 4 months of differentiation. Only modest adjustments of expression level of the NFκB (Table 1) levels were seen during differentiation while some components of the TLR signaling pathways (TICAM1, TICAM2 and IRAK1) were consistently increased (Table 2). A full list of gene expression changes during differentiation is found in Table S2.

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