A. I. Squillaro1,2, A. M. Fode2, L. A. Nucho2, S. M. Zuber1,2, D. F. Chang2, C. R. Schlieve1,2, T. C. Grikscheit1,2,3 1Children’s Hospital Los Angeles,Division Of Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine At The Saban Research Institute,Los Angeles, CA, USA 3University Of Southern California,Keck Medical School,Los Angeles, CA, USA
Introduction: Donor scarcity remains an obstacle for patients requiring orthotopic liver transplantation, although in some inborn errors of metabolism, replacement of an entire organ may not be necessary to prevent the buildup of toxic metabolites. Liver cell transplantation has been proposed for these patients; however, primary liver cells have limited expansion in vitro and still require donor tissue. Human induced pluripotent stem cells (hiPSC) offer a renewable source for cellular therapies that, unlike human embryonic stem cells (hESC), are derived from postnatal tissues. We initially derived hepatic progenitor cells from hESC after 17 days of directed differentiation. However, in order to plan to meet regulatory standards, we next hypothesized that hepatic progenitor cells (HPC) might be be generated from a fully characterized GMP hiPSC line, LiPSC-GR1.1, in a variation of our tested differentiation protocol.
Methods: LiPSC-GR 1.1 hiPSC were cultured in suspension for 17 days and differentiated into hepatospheres containing HPC. These hepatospheres were then analyzed by H&E and immunofluorescence staining for hepatic nuclear 4α (HNF4α), α- fetoprotein (AFP), and cytokeratin 19 (CK19), a marker for cholangiocytes. Immunofluorescent staining for proliferating cell nuclear antigen (PCNA) evaluated the presence of proliferation.
Results: H&E revealed formed hepatospheres. Immunofluorescence staining for hepatocyte markers HNF4α, an important regulator in metabolism, and AFP confirmed that hepatic progenitor cells were formed after 17 days of directed differentiation in vitro (Figure 1A). Cholangiocytes were detected in the HPC by the presence of CK19. Active proliferation of cholangiocytes were identified by PCNA co-staining (Figure 1B).
Conclusion: A fully characterized, GMP-compliant hiPSC line can produce hepatic progenitor cells after 17 days in vitro. The day 17 cells form hepatospheres that contain hepatocytes and cholangiocytes which are actively proliferating. Further investigation of hepatic progenitor cells from this source may provide a functional cellular therapy that salvages patients from congenital metabolic diseases.
