Assembly and Function of a Bioengineered Human Liver for Transplantation Generated Solely from Induced Pluripotent Stem Cells

Authors:
Kazuki Takeishi, Alexandra Collin de l’Hortet, Yang Wang, Kan Handa, Jorge Guzman-Lepe, Kentaro Matsubara, Kazutoyo Morita, Sae Jang, Nils Haep, Rodrigo M. Florentino, Fangchao Yuan, Ken Fukumitsu, Kimimasa Tobita, Wendell Sun, Jonatha Franks, Evan R. Delgado, Erik M. Shapiro, Nicolas A. Fraunhoffer, Andrew W. Duncan, Hiroshi Yagi, Tomoji Mashimo, Ira J. Fox, and Alejandro Soto-Gutierrez
In:
Source: Cell Rep
Publication Date: (2020)
Issue: :
Research Area:
Regenerative medicine
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector™ X-Unit
Experiment

- To test endothelial cell function, human neonatal microvascular endothelial cells (Lonza, Walkersville, MD) or human iPSC-VECs (iCell Endothelial Cells) (Cellular Dynamics, Fujifilm, Madison, WI) in culture (2D) and assembled vasculature of liver scaffolds were incubated in EBM PLUS Basal Medium supplemented with EGM-2 SingleQuots Kit (Lonza, Walkersville, MD) for human neonatal microvascular endothelial cells.

- Reprograming of fetal fibroblasts was performed using episomal plasmids vectors adapted from a previously described protocol (Okita et al., 2011). Briefly, for each nucleofection, 1 million cells were resuspended in 100 uL of the Amaxa TM NHDF Nucleofector kit (Lonza, Walkersville, MD), containing 3 ug of each of the four episomal plasmids vectors encoding OCT3/4 and p53 shRNA, SOX2 and KLF4, L-MYC and LIN28, and enhanced green fluorescent protein (eGFP) (Addgene, Boston, MA). Cells were nucleofected using the Amaxa 4D-Nucleofector (Lonza, Walkersville, MD) and plated in mTeSR1TM on human embryonic stem cell–qualified Matrigel (Corning, New York, NY)-coated plates.

- HCM bulletkit components were used in iPSC derived hepatocytes maturation stages.

Abstract

The availability of an autologous transplantable auxiliary liver would dramatically affect the treatment of liver disease. Assembly and function in vivo of a bioengineered human liver derived from induced pluripotent stem cells (iPSCs) has not been previously described. By improving methods for liver decellularization, recellularization, and differentiation of different liver cellular lineages of human iPSCs in an organ-like environment,we generated functional engineered human mini livers and performed transplantation in a rat model. Whereas previous studies recellularized liver scaffolds largely with rodent hepatocytes, we repopulated not only the parenchyma with human iPSC-hepatocytes but also the vascular system with human iPS-endothelial cells, and the bile duct network with human iPSC-biliary epithelial cells. The regenerated human iPSC-derived mini liver containing multiple cell types was tested in vivo and remained functional for 4 days after auxiliary liver transplantation in immunocompromised, engineered (IL2rg/) rats.