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Human embryonic stem cells (hESCs) can be progressively differentiated into definitive endoderm (DE), hepatic progenitors, and hepatocytes, and thus provide an excellent model system for the mechanistic study of hepatocyte differentiation, which is currently poorly understood. Here, we found that insulin enhanced hepatocyte differentiation from hESC-derived DE. Insulin activated the PI3K/AKT pathway, but not the mitogen-activated protein kinase pathway in the DE cells, and inhibition of the PI3K/AKT pathways by inhibitors markedly inhibited hepatocyte differentiation. In addition, insulin-like growth factor 1 (IGF1) and IGF2 also activated the PI3K/AKT pathway in DE cells and their expression was robustly upregulated during hepatocyte differentiation from DE. Furthermore, inhibition of IGF receptor 1 (IGF1R) by a small molecule inhibitor PPP or knockdown of the IGF1R by shRNA attenuated hepatocyte differentiation. Moreover, simultaneous knockdown of the IGF1R and the insulin receptor with shRNAs markedly reduced the activation of AKT and substantially impaired hepatocyte differentiation. The PI3K pathway specifically enhanced the expression of HNF1 and HNF4 to regulate hepatocyte differentiation from DE. Although inhibition of the PI3K pathway was previously shown to be required for the induction of DE from hESCs, our study revealed a positive role of the PI3K pathway in hepatocyte differentiation after the DE stage, and has advanced our understanding of hepatocyte cell fate determination.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133(+) HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.