Yonghak Kim

Generation of endoderm‐derived human induced pluripotent stem cells from primary hepatocytes

Recent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient‐specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm–derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm‐derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte‐derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency‐associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. Conclusion: The technology to develop endoderm–derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease‐specific iPS cells. (HEPATOLOGY 2010;51:1810–1819)

In Vivo Liver Regeneration Potential of Human Induced Pluripotent Stem Cells from Diverse Origins

Hepatic cells derived from human induced pluripotent stem cells of various origins contribute to liver regeneration in vivo. Treating Liver Disease, A Promethean Task As the ancient Greek legend of the disgraced Prometheus showed, the only human organ that can regenerate itself is the liver. Despite the liver’s remarkable capacity for repair and regeneration, diseases such as liver cirrhosis or hepatocellular carcinoma eventually destroy this ability and the only option is for patients to receive a liver transplant. But there is a severe shortage of donor livers for transplantation, which has prompted interest in stem cell therapy for treating patients with end-stage liver disease. However, liver stem cells are difficult to isolate and expand in culture so alternatives are being sought. Enter Liu et al. with a stem cell strategy that involves deriving mature human liver cells (hepatocytes) from human induced pluripotent stem cells (iPSCs). First, these investigators generated human iPSCs from a variety of adult human cells including hepatocytes, fibroblasts, and keratinocytes and showed that although these iPSCs were derived from very different cell types, they retained similar (although not identical) epigenetic signatures. The authors then used an established stepwise differentiation protocol to induce these human iPSCs to differentiate along the hepatic lineage first into definitive endoderm, then hepatic progenitor cells, and finally into mature hepatocyte-like cells. They found that, regardless of their origin, the different human iPSC lines all showed the same ability to differentiate into hepatic cells. To be useful for cell therapy, these human iPSC-derived hepatic cells must be able to engraft in liver tissue and function in the same way as normal human hepatocytes. So, the authors tested their human iPSC-derived hepatic cells (at different stages of differentiation) for their ability to engraft liver tissue in a xenograft model comprising immunodeficient mice treated with a chemical to induce liver injury. They intravenously infused the mice with 2 million human iPSC-derived hepatic cells or with normal human hepatocytes as a control. They found that human iPSC-derived hepatic cells engrafted mouse liver with an efficiency ranging from 8 to 15%, comparable to that for adult human hepatocytes (~11%). But were the engrafted human hepatic cells functional? The authors report that proteins normally secreted by adult human hepatocytes, such as albumin, transferrin, α-1-antitrypsin, and fibrinogen, could be detected in the serum of mice transplanted with human iPSC-derived hepatic cells at concentrations of 46, 101, 8.1, and 1100 ng/ml, respectively. Although preliminary, these encouraging findings suggest that it may be possible in the future to use infusions of human iPSC-derived hepatic cells to rescue injured liver tissue in patients with end-stage liver disease. Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.

Efficient drug screening and gene correction for treating liver disease using patient‐specific stem cells

Patient‐specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease‐specific iPSCs have been generated, there has been limited progress in iPSC‐based drug screening/discovery for liver diseases, and the low gene‐targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha‐1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease‐causing mutation with a high efficiency. A high‐throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96‐well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large‐scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC‐derived hepatocyte‐like cells. In addition, using the recently developed transcription activator‐like effector nuclease technology, we achieved high gene‐targeting efficiency in AAT‐deficiency patient iPSCs with 25%‐33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte‐like cells derived from the gene‐corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost‐effective targeting technology will broadly benefit both basic and translational applications. Conclusions: Our results demonstrated the feasibility of effective large‐scale drug screening using an iPSC‐based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. (HEPATOLOGY 2013;57:2458–2468)

Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

EBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line-derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies.

Capsiate, a Nonpungent Capsaicin-Like Compound, Inhibits Angiogenesis and Vascular Permeability via a Direct Inhibition of Src Kinase Activity

Capsiate, a nonpungent capsaicin analogue, and its dihydroderivative dihydrocapsiate are the major capsaicinoids of the nonpungent red pepper cultivar CH-19 Sweet. In this study, we report the biological actions and underlying molecular mechanisms of capsiate on angiogenesis and vascular permeability. In vitro, capsiate and dihydrocapsiate inhibited vascular endothelial growth factor (VEGF)-induced proliferation, chemotactic motility, and capillary-like tube formation of primary cultured human endothelial cells. They also inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessels in the mouse Matrigel plug assay in response to VEGF. Moreover, both compounds blocked VEGF-induced endothelial permeability and loss of vascular endothelial (VE)-cadherin-facilitated endothelial cell-cell junctions. Importantly, capsiate suppressed VEGF-induced activation of Src kinase and phosphorylation of its downstream substrates, such as p125(FAK) and VE-cadherin, without affecting autophosphorylation of the VEGF receptor KDR/Flk-1. In vitro kinase assay and molecular modeling studies revealed that capsiate inhibits Src kinase activity via its preferential docking to the ATP-binding site of Src kinase. Taken together, these results suggest that capsiate could be useful for blocking pathologic angiogenesis and vascular permeability caused by VEGF.

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.

IFNL3 genotype is associated with differential induction of IFNL3 in primary human hepatocytes

BACKGROUND Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive. METHODS IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3. RESULTS PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07). CONCLUSIONS Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection.

Drug Discovery Using Human iPSC Based Disease Models and Functional Hepatic Cells

The development of human induced pluripotent stem cell (iPSC) technology has generated enthusiasm about the therapeutic potential of these cells for treating a variety of diseases. During the past few years, iPSC generation and hepatic differentiation methods have been significantly improved. These will provide an unlimited source of functional hepatocytes not only for transplantation, but for efficient drug discovery via patient relevant modeling of liver diseases and of drug-induced hepatotoxicity. Here we discuss the near future applications (and challenges) of iPSC-based cellular models, with an emphasis on liver diseases, cancer and hepatocytes.