Professor of Surgery, Harvard Medical School
Chief, Division of Transplantation, Beth Israel Deaconess Medical Center
Effects of interlacing endothelial cells on in-vitro differentiation of induced pluripotent stem cell embryoid bodies into hepatocyte-like cells
Giuseppe Pettinato1,2,3, Rajesh Ramanathan4, Martin J Mangino4, Xuejun Wen3, Robert A Fisher1,2.
1Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA, United States; 2Department of Surgery, Harvard Medical School, Boston, MA, United States; 3Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, United States; 4Department of Surgery, Virginia Commonwealth University Medical Center, Richmond, VA, United States
Introduction: The scalable, efficient and durable differentiation of human induced pluripotent stem cells (hiPSC) into functional hepatocyte-like cells has applications in cell therapy, bioengineered organs and pharmaceutical testing. This study used a novel suspension-based embryoid body differentiation protocol for the generation of iPSC-derived hepatocyte-like cells (HLC) and tested the effect of co-culture with human endothelial cells on improving function and longevity after transplantation in an animal model of acute liver failure.
Methods: Embryoid bodies with hiPSC only (hiPSC-EB) and containing hiPSC mixed with endothelial cells (hiPSC+EC-EB) were generated using agarose molds. hiPSC-EB and hiPSC+EC-EB were differentiated into hepatocyte-like cells using a four-stage suspension culture-based differentiation protocol. Both hiPSC-EB-HLC and hiPSC+EC-EB-HLC were screened for several hepatic functionalities and markers in vitro and transplanted into the spleens of athymic rats with d-galactosamine induced acute liver failure.
Results: The differentiation of hiPSC-EB and hiPSC+EC-EB into hepatocyte-like cells was confirmed by the presence of gene expression and immunofluorescence for several hepatocyte markers such as Albumin, C-Met, CK-18 and HNF-1 α and several CYP450 isoforms. hiPSC+EC-EB-HLC showed an increased amount of Albumin secretion in vitro compared to the hiPSC-EB-HLC. hiPSC+EC-EB-HLC displayed higher secretion of Alpha-Fetoprotein, Fibrinogen and Urea as compared to the hiPSC-EB-HLC. Hepatocyte function in vitro, such as Acetylated low-density lipoprotein (DiI-ac-LDL) uptake, Indocyanine green (ICG - Cardiogreen) absorption and release after 6 hours, Glycogen storage, and cytoplasmic accumulation of neutral triglycerides and lipids were comparable between hiPSC+EC-EB-HLC and hiPSC-EB-HLC. The induction of several cytochromes P450 (CYP450) through the use of different inducers demonstrated a significant augmentation in the activity of all the CYP450 tested from baseline for the hiPSC+EC-EB-HLC in comparison to the hiPSC-EB-HLC. Transplantation of hiPSC+EC-EB-HLC was associated with sustained rat serum human albumin at 14 days after transplant as compared to 3 days after transplantation among the hiPSC-EB-HLC group. The mean survival of the transplanted rats was comparable between the two experimental conditions.
Conclusion: Embryoid bodies are a viable and scalable means to produce functional hiPSC-derived hepatocyte-like cells. The incorporation of endothelial cells with hiPSC in embryoid bodies provides more sustained hepatocyte function in vivo after transplantation.
13:30 - 15:00
|Roadblocks in Clinical Translation||Problems with hepatocytes||Room 111-112|
15:30 - 17:00
|Stem Cells Bioengineering, and Preclinical Work||Effects of interlacing endothelial cells on in-vitro differentiation of induced pluripotent stem cell embryoid bodies into hepatocyte-like cells||Room 111-112|
11:00 - 12:30
|Tissue Engineering and Stem Cell Differentiation||Co-culture with primary liver cells improves maturation and differentiation of Human Induced Pluripotent Stem Cells-derived hepatocyte-like cells||Room 111-112|