Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition.
Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1.
Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA.|Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.|Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, USA.
Giuseppe
G
Pettinato
Department of Surgery, Virginia Commonwealth University Medical Center, Richmond, VA, USA.
Rajesh
R
Ramanathan
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Robert A
RA
Fisher
Department of Surgery, Virginia Commonwealth University Medical Center, Richmond, VA, USA.
Martin J
MJ
Mangino
Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, USA.
Ning
N
Zhang
Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA.|Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, Shanghai 200120, People's Republic of China.
Xuejun
X
Wen
D048868:Adaptor Proteins, Signal Transducing / Q000494:pharmacology*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D004195:Disease Models, Animal; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D017114:Liver Failure, Acute / Q000378:metabolism / Q000628:therapy*; D051381:Rats; D012097:Repressor Proteins / Q000494:pharmacology*; D000075462:Serum Albumin, Human / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D012097:Repressor Proteins; C485569:WIF1 protein, human; D000075462:Serum Albumin, Human