(122b) Optmization of Intermediate Steps of the Beta Islet Differentiation Program for Human Embryonic Stem Cells

Differentiation of human embryonic stem cells (hESC) into insulin expressing cells has been achieved by several groups with some limitations. A multiple step differentiation protocol is needed in order to obtain cells that resemble pancreatic Beta islets. The first step of this multi-step approach is differentiation of stem cells into definitive endoderm (DE) cells. It is believed that in order to achieve pancreatic differentiation, each step of the differentiation program has to be manipulated to obtain cells that closely mimic the different stages of embryogenesis. The objective of this study is to optimize differentiation of human embryonic cells into DE cells that meet a defined criterion set by current knowledge of pancreatic organogenesis.

Several groups have developed protocols that use Activin A in combination with different growth factors to achieve endodermal differentiation. Enoderm differentiation however is a complex system due to the existence of different endodermal derivatives that have similar marker expression patterns but result in formation of very different tissues. Visceral endoderm (VE) for instance results in formation of extra-embryonic tissue but shares many of the same expression hallmarks as DE. CXCR4 is a membrane marker that has previously been found to be expressed in mesoderm and DE but not in VE. SOX17 on the other hand is expressed in both VE and DE but not in mesoderm, while Brachyry T is expressed in mesoderm but not endoderm derivatives. Based on this we want to find cells that express high levels of CXCR4 expression and co-expression, but low levels of Brachyury T.

This study looks at the transcriptional regulatory networks in which FGF2, BMP4, WNT3A and PI3 Kinase inhibitor (PI3KI) participate during differentiation of human embryonic stem cells into DE cells. We found all factors to induce a significant upregulation of both CXCR4 and SOX17 when compared to undifferentiated hESC, while Brachyury T was downregulated. These suggest that all factors induce differentiation into endodermal derivatives. Given the downregulation of Brachyury, CXCR4 alone becomes a good representation of DE, while SOX17 gives us information about other endodermal derivatives. The highest CXCR4 upregulation for individual factors was found to be achieved by PI3KI by a factor of 3 to 4, while BMP4 showed the lowest. FGF2 and BMP4 however showed a synergistic effect and when used in combination showed CXCR4 upregulation similar to that found in PI3KI, conversely, WNT3A appeared to have an antagonistic effect on BMP4 with the lowest upregulation of CXCR4 achieved of all groups. When differentiated with BMP4, FGF2 and WNT3A, the level of CXCR4 upregulation was higher than the upregulation found by each factor alone, suggesting that the synergistic effect of FGF2 on BMP4 is stronger than the antagonistic effect exerted by WNT3A. BMP4 also showed to have a high upregulation of SOX17, which suggests that it induces differentiation of other endoderm derivatives aside from DE. Interestingly, the factors and combinations that resulted in highest upregulation of CXCR4 also had the highest upregulation of SOX17 which could be suggestive of co-expression of these markers, therefore confirmation that cells differentiate mostly into DE cells.

An in depth analysis of the results obtained in these study gives us an insight of the transcriptional networks governing DE differentiation and the interactions and effects that different pathways have. It will allow us to develop models that will aid the optimization of different steps of Beta Islet differentiation to obtain cells with phenotypes that closely resembles that of native pancreatic cells.