Generation of safe and therapeutically effective human induced pluripotent stem cell‐derived hepatocyte‐like cells for regenerative medicine

Abstract

Hepatocyte‐like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. First, human iPS‐HLCs were generated from a human leukocyte antigen‐homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder‐free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1‐positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS‐HLC transplantation. Furthermore, harmful genetic somatic single‐nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast‐like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS‐HLCs, these cells (1 × 106 cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl4 only once and chronic liver injury mice treated with 0.6 mL/kg CCl4 twice weekly for 8 weeks. By human iPS‐HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased. Conclusion: We were able to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. (Hepatology Communications 2017;1:1058–1069)

Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells‐derived hepatic stem cells

“…Recent studies described the production of transplantable HLCs using GMP‐compatible hepatic differentiation protocol, but culture times were about 30 days vs 11 days for pHSCs. Takayama et al demonstrated a very efficient cryopreservation of HLCs using a GMP‐compatible cryopreservation solution, but cells functionality was not confirmed in vivo.…”

“…Recent studies described the production of transplantable HLCs using GMP‐compatible hepatic differentiation protocol, but culture times were about 30 days vs 11 days for pHSCs. Takayama et al demonstrated a very efficient cryopreservation of HLCs using a GMP‐compatible cryopreservation solution, but cells functionality was not confirmed in vivo. While our study did not allow to compare pHSCs efficiency with the one of HLCs, our data show that pHSCs can efficiently be transplanted in Gunn rats to correct hyperbilirubinemia, using a protocol asking two times less time, and in absence of liver pre‐conditioning that would give a selective growth advantage to transplanted hepatocytes.…”

Analysis of the mechanism underlying liver diseases using human induced pluripotent stem cells

“…Several reports have shown that human iPS cells differentiate into hepatocyte or cholangiocyte lineages in vitro [4][5][6][7][8]. Hepatocyte-like cells derived from iPS cells (iPS-Heps) exhibit properties of hepatocytes, such as albumin secretion and metabolic functional properties.…”

“…Human induced pluripotent stem (iPS) cells are somatic cells genetically reprogrammed to be pluripotent by the transient expression of genes essential for maintaining the properties of embryonic stem cells [3]. Human iPS and human embryonic stem cells exhibit the potential for differentiation into various cell types, including hepatic lineages [4][5][6][7][8]. Such pluripotent stem cells are candidates for regenerative medicine and a promising tool to evaluate the pathophysiology of genetic or non-genetic diseases.…”

Tolloid‐Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling

“…Human pluripotent stem cells are useful as a model of liver development because they differentiate into hepatocytes by mimicking early liver development. We optimized the growth factors and small molecular compounds added in hepatic differentiation and succeeded in developing an efficient hepatic differentiation protocol of human induced pluripotent stem (iPS) cells . In addition, we searched for genes and compounds that can improve the homologous recombination efficiency of human iPS cells using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas9 system.…”