Yoon Young Jang

Hematopoietic stem cells convert into liver cells within days without fusion.

Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2–7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.

Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34(+) cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34(+) cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34(+)CD45(+)) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34(+) cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

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)

Efficient and Allele-Specific Genome Editing of Disease Loci in Human iPSCs

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

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.

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.

Pluripotent Stem Cell-Based Cancer Therapy: Promise and Challenges

Many technical hurdles must be surmounted before induced pluripotent stem cells can be used in cancer therapy. The development of induced pluripotent stem cell (iPSC) technology has generated enthusiasm about the therapeutic potential of these cells for treating a variety of diseases. However, the evidence that they actually will be clinically useful is limited. Here, we discuss the potential therapeutic applications of iPSCs for treating cancer and other diseases and highlight the current barriers restricting their use.

Stem cell plasticity: a rare cell, not a rare event.

Purification to homogeneity for a rare stem cell (SC) population by both function and phenotype is a prerequisite to determine if SCs can change their fate (plasticity). Since cell fate determination has been suggested by both external environmental cues and intrinsic gene regulation, plasticity should be studied using both influences. Different frequencies of marrow SC plasticity may be attributed to either different isolation technologies or different developmental stage SCs with more or less multipotentiality. Tissue-specific SCs may reside in marrow, or alternatively, primitive marrow SC may respond directly to regenerative signals by migration to injury sites and repairing the damaged tissue. It is important to dissect the relationship between primitive/tissue-specific SCs and regenerative signals.