Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Abstract

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

“…The minimal ureagenesis observed in the Arg1 Δ hepatocytes is likely residual expression resulting from incomplete tamoxifen-mediated removal of exons 7 and 8. Nevertheless, our observations are in accordance with a previous report that showed consistent expression of Afp accompanied by low Arg1 expression and urea synthesis at the final stage of differentiation relative to primary hepatocytes 29 . To evaluate the expression of other urea cycle enzymes, we performed quantitative real-time PCR (qRT-PCR) and observed scarce expression of the five main ureagenic enzymes, providing further evidence of incomplete maturation (Fig.…”

“…4d). These data suggested that either the cells were not mature enough for high-level Arg1 expression by the chosen differentiation protocol 29 and/or that there were problems with maturation of the RFP fluorophore (see Discussion).

Figure 4Generation of hepatocyte-like cells from repaired mouse iPSCs. ( a ) Schematic diagram showing the stepwise hepatic differentiation protocol.

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“…Our ongoing efforts include refining our current hepatic differentiation protocol, in particular the final step of differentiation to acquire a more mature phenotype. In vivo transplantations of both human and mouse immature iHLCs promote further maturation and long-term repopulation of hepatocytes 29, 43 . Thus, it is possible that the transplantation of gene-corrected iHLCs into our mouse model could enhance functional maturation.…”

Engineering a Tumor Microenvironment‐Mimetic Niche for Tissue Regeneration with Xenogeneic Cancer Cells

“…All of them grew normally in THAG but not in ACG (Figure S7, Supporting Information). Remarkable epithelial morphogenesis was recapitulated in the neo‐tissues formed in the cell‐laden THAG, where Caco2 and Hep G2 cells developed into tubal organoids ( Figure 5 a,c, Figures S8 and S9, Supporting Information) 35, 36. IF co‐staining for human retinol‐binding protein II (RBP 2) or human hepatic nuclear factor 4 alpha (HNF 4α)—markers of Caco2 and Hep G2 cells,34, 37 respectively—and E‐cadherin further testified that the tubular structures were constituted by the formed epithelium organoids (Figure 5b and d).…”

High Efficient Differentiation of Functional Hepatocytes from Porcine Induced Pluripotent Stem Cells

“…Although a variety of hepatic differentiation methods have been established in mouse and human iPSCs [14], [15], [16], less attention has been paid to iPSC-derived from large animals such as pigs. The efficent production of hepacocytes from iPCSs is heavily reliant on the deep understanding of early stage hepatogenesis.…”