Human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and the technology to developmentally program these cells to various cell lineages offer great promise for cell therapy of various diseases. Recently, different protocols to differentiate them into hepatocytes-like cells (HLC) have been described. In this context, we hypothesize that this approach could be useful in developing a relevant model for HCV infection in vitro. Moreover, the hiPSCs approach could allow the generation of patient-specific hepatocytes with promising opportunity for cell therapy of viral liver diseases. We have generated hiPSCs from primary fibroblasts using lentiviruses or Sendai virus vectors, and characterized them in comparison to hESCs. Human pluripotent stem cells were efficiently differentiated into HLC, as demonstrated by induction of the expression of hepatic markers and the secretion of hepatic proteins (AFP and albumin) in the supernatants. Moreover these cells recapitulate hepatocyte-specific metabolic functions like lipid and glycogen accumulation, and indocyanin green metabolism. We also successfully engrafted, via intra-splenic injection, 2-4 millions HLCs into the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter (MUP-uPA/SCID/Bg). In collaboration with Dr. Cindy Dunbar's lab, we have also extended the hepatic differentiation protocol to monkey iPSC lines. The goal is to establish a monkey model for potential stem cell-based therapy. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. We have developed a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain highly differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSCs-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli. In collaboration with NCATS, we are currently screening small-molecule libraries to identify compounds that may regulate hepatic differentiation. HBV and HCV are both blood borne and hepatotropic pathogens. In patients with HBV-HCV coinfection, HBV reactivation leading to severe hepatitis has been reported with the use of interferon-free direct-acting antiviral agents to treat HCV infection. Here we study the molecular mechanisms leading to HBV reactivation after HCV elimination in HBV-HCV coinfection. HBV-HCV coinfection was first established in primary human hepatocytes (PHHs). HBV replication was suppressed by HCV infection as compared to HBV monoinfection. This suppression could be partially rescued by sofosbuvir and fully rescued by inhibitor of Janus kinase (Jaki), which results in complete blocking of Interferon-stimulated genes induced by HCV infection. Both treatments had no effect on HBV monoinfection. In contrast, HCV infection was not affected by HBV co-infection or enticavir treatment of co-infected cells. Analyses at the single cell level by in situ hybridization (RNAscope) validated that HBV nucleic acid level was greatly reduced in both HCV positive cells and negative cells after HCV infection. Jaki treatment could restore HBV nucleic acid level in coinfected cells to the level in HBV monoinfection, consistent with the role of HCV-induced interferon response in suppressing HBV replication. To further dissect the mechanism of possible viral interference between HBV and HCV, PHH-transplanted Alb-UPA/SCID mice were analyzed. HBV viremia was significantly lower in HBV-HCV coinfected mice than in HBV monoinfected mice. Treatmet of co-infected mice with DAA or entecavir is currently under investigation to address the question of whether or not HCV elimination by DAA could lead to HBV reactivation in the coinfected mice as well as the underlying innate immune mechanisms of viral interference. In conclusion, we found HCV infection suppresses HBV infection in PHHs in vitro as well as in Alb-UPA/SCID mice in vivo. This suppression is likely due to the interferon responses induced by HCV infection instead of direct interference between the two viral replicative pathways, possibly explaining the clinical observation of potential HBV reactivation in DAA-treated patients with HBV-HCV co-infection.
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