Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.     

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.     

Cholesterol-Secreting and Statin-Responsive Hepatocytes from Human ES and iPS Cells to Model Hepatic Involvement in Cardiovascular Health

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis, and their proper function is of key importance for cardiovascular health. In particular, hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis, dyslipidemias, the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol, actively secrete cholesterol into the medium, and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly, it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally, the system will also be useful for mechanistic studies of heritable dyslipidemias, drug discovery, and investigation of modes of action of cholesterol-modulatory drugs.     

The road to regenerative liver therapies: The triumphs,trials and tribulations

The liver is one of the few organs that possess a high capacity to regenerate after liver failure or liver damage. The parenchymal cells of the liver, hepatocytes, contribute to the majority of the regeneration process. Thus, hepatocyte transplantation presents an alternative method to treating liver damage. However, shortage of hepatocytes and difficulties in maintaining primary hepatocytes still remain key obstacles that researchers must overcome before hepatocyte transplantation can be used in clinical practice. The unique properties of pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) have provided an alternative approach to generating enough functional hepatocytes for cellular therapy. In this review, we will present a brief overview on the current state of hepatocyte differentiation from PSCs and iPSCs. Studies of liver regenerative processes using different cell sources (adult liver stem cells, hepatoblasts, hepatic progenitor cells, etc.) will be described in detail as well as how this knowledge can be applied towards optimizing culture conditions for the maintenance and differentiation of these cells towards hepatocytes. As the outlook of stem cell-derived therapy begins to look more plausible, researchers will need to address the challenges we must overcome in order to translate stem cell research to clinical applications.     

Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.     

Induced pluripotent stem cells: a new technology to study human diseases

Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed to a pluripotent state by the introduction of specific factors. They can be generated from cells of different origins such as fibroblasts, keratinocytes, hepatocytes and blood. iPS cells are similar to embryonic stem cells in several aspects such as morphology, expression of pluripotency markers and the capacity to develop teratomas; tumors containing cells of the three germ layers. As pluripotent stem cells they can be differentiated into several lineages including neuronal, cardiac and blood cells. Recently, several groups have successfully generated patient-specific iPS cells from donors suffering different disorders and differentiated them into the cell type affected by the disease. These new human cell-based models cannot only be used to study the dynamics of diseases but also as systems to screen new drugs. Moreover, iPS cells promise to be good candidates for regenerative medicine.     

The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Background

Liver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).

Methods/Principal Findings

Mice received CCl₄ injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl₄ injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl₄-injuries.

Conclusions/Significance

These findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.     

Are induced pluripotent stem cells the future of cell‐based regenerative therapies for spinal cord injury?

Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited. During the last two decades, the search for new therapies has been revolutionized by the discovery of stem cells, inspiring scientists and clinicians to search for stem cell‐based reparative approaches for many disorders, including neurotrauma. Cell‐based therapies using embryonic and adult stem cells in animal models of these disorders have provided positive outcome results. However, the availability of clinically suitable cell sources for human application has been hindered by both technical and ethical issues. The recent discovery of induced pluripotent stem (iPS) cells holds the potential to revolutionize the field of regenerative medicine by offering the option of autologous transplantation, thus eliminating the issue of host rejection. Herein, we will provide the rationale for the use of iPS cells in SCI therapies. In this review, we will evaluate the recent advancements in the field of iPS cells including their capacity for differentiation toward neural lineages that may allow iPS cells transplantation in cell‐based therapy for spinal cord repair. J. Cell. Physiol. 222: 515–521, 2010. © 2009 Wiley‐Liss, Inc.     

Hepatic differentiation of adult and fetal liver stromal cells in vitro

The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. For example, in patients with alcoholic cirrhosis repair mechanisms are not activated and only organ transplantation or advanced methods of regenerative medicine can help such patients. Clinical trials including patients with various forms of liver disease have shown promising results of transplantation of autologous bone marrow stem cells. However, improvement of the effectiveness of such treatment requires optimization of sources of progenitor cells. In this study we have isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, performed their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. Stromal cells isolated from the fetal liver were used for comparative evaluation. During hepatic differentiation both types of cells expressed hepatic markers and secreted albumin. These results can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies proliferate for a long time in a culture and this provides opportunity to produce them in large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.     

利用piggyBac转座子制备牛成体多能干细胞诱导技术研究

通过逆转录病毒等媒介表达核转录因子Oct4、Sox2、Klf4、c-Myc可将体细胞重编程为诱导多能干细胞(induced pluripotent stem cells, iPSc)。时至今日,已经报道了小鼠、人、大鼠、猪、羊、马、牛的iPS细胞,但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认。与逆转录病毒等不同的是,piggyBac转座子转染效率高且无病毒源性、操作简单,可以在转座酶的存在下被安全切除。首次尝试了采用piggyBac转座子携带鼠源Oct4、Sox2、Klf4、c-Myc、Rarg和Lrh16个核转录因子诱导胎牛成纤维细胞,成功获得牛类iPS细胞,其形态与小鼠胚胎干细胞相似,克隆边界清晰、呈丘状、克隆内细胞致密、核大。RT-PCR与免疫组织化学染色分析均显示牛类iPS细胞表达多能性基因。该类细胞体外诱导分化可形成类胚体EB,且表达3个胚层的基因;体内诱导分化可形成畸胎瘤,苏木精、伊红染色显示瘤体有三胚层的分化。上述结果显示利用piggyBac转座子制备牛多潜能干细胞诱导技术可行,产生的牛类iPS细胞具有潜在多能性。     

Commitment of stem cells into functional hepatocytes

Liver transplants represent the only way to treat patients suffering from terminal liver failure, but they are associated with numerous problems, including a chronic shortage of donors, high cost, rejection, and side effects for the donor. It is anticipated that regenerative medicine will provide an alternative to liver transplants for such patients. Regenerative medicine refers to the academic field of eliciting the inherent capacity of organisms for self-regeneration to the greatest possible extent in order to develop new methods of treatment for intractable disorders. From this perspective, much is expected from the use of human embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells), and the vigorous development of technology to induce the differentiation of such stem cells into cells possessing hepatic functions is underway. Clinical applications of these human stem cells, however, have yet to reach even the earliest stages of implementation. Facing off against these versatile ES cells are stem cells derived from somatic cells present within organisms, which are attracting attention owing to their superiority in terms of ethics and safety, with many research institutes now in the process of elucidating the details of stem cell separation and identification as well as their plasticity and pluripotency. Bone marrow cells are the best-known somatic-cell-derived stem cells, but the use of mesenchymal stem cells (MSCs) found in adipose tissue has also recently attracted attention. This paper will review the differentiation ability and mechanisms of these various stem cell types to hepatocytes and their application to liver regeneration and the future outlook.     

疾病特异诱导多能干细胞研究进展

通过病毒或非病毒转导体系,在小鼠和人的体细胞中人为表达几个与细胞多能性相关的转录因子,从而使细胞达到类似于胚胎干细胞（embryonic stem cells,ESCs）状态,是近年来新发展起来的体细胞重编程技术。这些被重编程的细胞称为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。这项技术为获得患者和疾病特异的多能干细胞提供了新的途径。患者和疾病特异的iPS细胞的获得,不仅在避免免疫排斥的宿主特异的细胞移植治疗上有广泛前景,并对了解疾病发生机理、药物筛选和毒性研究有着重要的意义。该文综述从iPS细胞技术的发明入手,着重讨论疾病iPS细胞的研究进展及其在应用于治疗时亟需解决的问题。     

Could co‐transplantation of iPS cells derived hepatocytes and MSCs cure end‐stage liver disease?

Orthotopic liver transplantation is, to date, the only proven effective treatment for end-stage liver disease. However, it suffers from lack of donors and immunorejection. Here, we speculate that co-transplantation of induced pluripotent stem (iPS) cells derived hepatocytes and mesenchymal stem cells (MSCs) may offer an alternative way to treat patients with end-stage liver disease. Recently, progress on iPS cells, homogeneous differentiation of hepatocyte-like cells from embryonic stem cells (ESCs), and paracrine effects by MSCs highlight the possibility. Safe, efficient and rapid generation of iPS cells has been reliably produced by several experimental laboratories. Methods for highly efficient and homogeneous differentiation of ESCs into functional hepatocytes have been established as well. Moreover, paracrine effects by MSCs have been proven to play an important role in liver regeneration and repair, and the effects can be used as an enhancer for engraftment. All of these remarkable developments lead to this hypothesis which may offer a novel therapeutic strategy for treatment of patients with end-stage liver disease, though some issues about safety and efficacy need to be further guaranteed.     

Generation,characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013. © 2012 Wiley Periodicals, Inc.