Induced pluripotent stem cells and hepatic differentiation : Journal of the Chinese Medical Association

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Review Article

Induced pluripotent stem cells and hepatic differentiation

Chiang, Chih-Hunga,b; Huo, Teh-Iaa,c; Sun, Cho-Chinb; Hsieh, Jung-Hungb; Chien, Yuehe; Lu, Kai-Hsid,e,f,*; Lee, Shou-Donge,g

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Journal of the Chinese Medical Association 76(11):p 599-605, November 2013. | DOI: 10.1016/j.jcma.2013.07.007

    Abstract

    1. Introduction

    According to the theory of ability differentiation, stem cells can be classified into three categories. Totipotent stem cells, which can be implanted in the uterus of a living animal and give rise to a full organism, belong to the first category. Pluripotent stem cells, which can give rise to every cell of an organism except extraembryonic tissues, belong to the second category, and this limitation is restricted to develop into a full organism. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are typical types of pluripotent stem cells. Multipotent stem cells, which are adult stem cells and only generate specific lineages of cells, belong to the third category.

    ESCs are pluripotent stem cells derived from the inner cell mass of mammalian blastocysts. Due to their remarkable ability to proliferate indefinitely under appropriate in vitro culture systems and their ability to differentiate into any cell types of all three germ layers, differentiation of ESCs in vitro provides a powerful model system for addressing questions related to lineage commitment.1,2 Since human ESCs were first isolated in 1998,3 ESCs have been regarded as a powerful platform or tool in developmental studies, drug screening, diseases treatment, tissue repair engineering, and regenerative medicine. However, two main limitations have impeded the application of ESC-based therapy. The first is the ethical dilemma regarding human embryo donation and destruction, and the second is that ESCs are incompatible with the immune system of patients. To circumvent these deficiencies, scientists worldwide have been devoted to the development of a variety of reprogramming techniques to reverse somatic cells into a stem cell-like state.

    In 2006, Dr. Shinya Yamanaka made a landmark discovery that reprogramming somatic cells back to iPSCs could be achieved by retroviral transduction of four pluripotency-associated transcription factors—octamer-binding transcription factor 4 (Oct3/4), sex determining region Y-box 2 (Sox2, SRY), V-myc myelocytomatosis viral oncogene homolog (c-Myc), and Kruppel-like factor 4 (Klf4).4 These iPSCs possessed morphological and molecular features that resemble those of ESCs. They also gave rise to teratoma and germline-competent chimeras upon injection into blastocysts. This amazing finding showed that cell fate could be manipulated by certain genes, and the discovery led to several distinguished awards, including the Albert Lasker Basic Medical Research Award in 2009, and the International Balzan Prize in 2010. In 2012, Dr. Shinya Yamanaka and John Gurdon were awarded the Nobel Prize for Physiology or Medicine for the discovery that mature cells can be converted to stem cells. iPSCs have been generated in various ways, including the exogenous gene delivery method,5–9 choosing multiple somatic cell sources,10–15 and induction of iPSCs by small compounds16 to improve the efficiency of the reprogramming process.

    2. Comparison of iPSCs with ESCs

    Generally, fully reprogrammed iPSCs display numerous properties similar to those of ESCs. First, iPSCs are morphologically identical to ESCs, and show infinite proliferation and self-renewal abilities. They express pluripotency markers such as TRA-1-60 antigens (TRA-1-60), TRA-1-80 antigens (TRA-1-80), stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2, SRY), and Nanog homeobox (Nanog).17–23 Typically, TRA-1-60 and SSEA-4 are used as selection markers to distinguish fully reprogrammed nascent colonies from partially reprogrammed ones. In addition, iPSCs pass the hallmark test of pluripotency; when injected into the testes of immunocompromised mice, they form teratomas, showing their potential to differentiate into the three embryonic germ layers. Moreover, the iPSCs also contribute to germ line transmission.24 Several molecular and functional assays were set to evaluate the similarity of iPSCs to ESCs, including reactivation of self-renewal and pluripotency associated genes, telomerase activity, X chromosome, and stage-specific embryonic surface antigens, suppression of somatic genes associated with cell of origin, silencing of exogenous factors, capabilities in in vitro differentiation, demethylation of promoters of pluripotency genes, and in vivo teratoma formation, chimera contribution, germline transmission, and tetraploid complementation.6,25,26 A recent study demonstrated that patient-specific iPSCs from dermal fibroblasts of patients with long QT syndrome can differentiate into functional cardiac myocytes, but still recapitulated the electrophysiological features of the disorder.27 Therefore, the major advantage of iPSCs over ESCs, is that iPSCs can be derived from a patient's own somatic cells, thereby avoiding immune rejection after transplantation and the ethical concerns raised by using ESCs.

    3. Advances in reprogramming techniques

    Based on their pluripotency, and that they are capable of differentiating into any functional cell type, iPSCs possess great potential for regenerative and therapeutic applications. However, the group led by Dr. Yamanaka also reported that those chimeras derived from mouse iPSCs, and their progeny often develop tumors, mainly due to reactivation of c-Myc transgene.28 Thus, numerous approaches to generate iPSCs with lower tumorigenicity have been developed. Several studies have shown that iPSCs generated without the c-Myc virus demonstrated reduced tumor incidence in chimeric mice, but the efficiency of iPSC creation is significantly reduced.29,30 To overcome this dilemma, Okita et al found another member of Myc, L-Myc, possessed stronger activity to generate iPS and less tumorigenic activity.32

    The use of genome-integrating retroviruses that are closely related to tumor formation was another major limitation of the original iPSC generation techniques. Thus, reprogramming strategies with non-integrating systems seem to be the solution to make iPSC-based therapy feasible. In 2008, Stadtfeld et al established mouse iPSCs from fibroblasts and liver cells, by non-integrating adenoviruses carrying four defined factors, and suggested that insertional mutagenesis is not required for in vitro reprogramming.31 At the same time, Okita et al successfully generated iPSCs by transient transfection of two plasmids containing complementary DNAs (cDNAs) encoding four factors, eliminating the transgenic integration by the use of retroviruses.32 More recent studies further described a “stem cell cassette” or a polycistronic virus, a single lentiviral vector composed of all four factors, and were able to yield iPSCs with reduced insertional mutagenesis and viral reactivation.33,34 Another novel reprogramming technique using piggyBac transposon was published in 2009.8,9,35 A polycistronic plasmid harboring four factors and piggyBac transposon was constructed and integrated into the genome in the presence of piggyBac transposase. As the reprogramming process was achieved, the inserted fragment was easily removed by re-expressing transposase. The transposon-based method eliminates the use of virus, displays equivalent efficiencies to retroviral transduction, excises integrated sequences without genome alteration, and therefore represents a landmark progress toward therapeutically relevant virus-free iPSCs. To avoid introducing exogenous genetic materials, two significant advances were reported. Zhou et al demonstrated that mouse fibroblasts could be fully reprogrammed by direct delivery of recombinant reprogramming proteins.36 In 2010, an impressive study by Luigi Warren and his colleagues showed a strategy for reprogramming by administering synthetic mRNAs that code for key factors, and by creating RNA-iPS (RiPS) cells.37 Both techniques are safer, simpler, and faster approaches than the currently established genetic method.

    In human livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, and hepatic progenitor cells are activated only when the mature epithelial cells of the liver are continuously damaged or in cases of severe cell loss, or inhibited in their replication.38,39 Stem cell niche refers to a microenvironment where stem cells are found, which interacts with stem cells to regulate cell fate and self-renewal. The interaction with the specific microenvironmental cells is thought to be a key mechanism in regulating the maintenance of self-renewal and differentiation capacities by stem cells. In the liver, the niche of hepatic progenitor cells is located at the level of the canals of Hering and is composed of numerous cells, such as hepatocytes, endothelial cells, hepatic stellate cells, cholangiocytes, Kupffer cells, and inflammatory cells. It has been reported that the autocrine and paracrine of Wnt secretion and growth factors could interact and cross-talk with progenitor cells, influence their proliferative and differentiate processes in mice, rats, and humans.40,41

    4. Advances in cell-based therapy

    The development of stem cell studies makes cell transplantation a promising therapy for diseases of the central nervous system (CNS), including stroke, traumatic brain injury, hypoxic encephalopathy, and degenerative disorders.42 Parkinson's disease (PD) is the best candidate for cell replacement therapy because only one group of cells, the dopaminergic neurons, are affected. The main pathology of PD is cellular loss of the substantia nigra pars compacta dopaminergic neurons that project to the striatum.43 Clinical signs of PD, which include rest tremor, rigidity, and bradykinesia, are evident when about 80% of striatal and 50% of nigral neurons are lost.44 Cell replacement therapy was first attempted using fetal mesencephalic tissue, and the results were successful in the earliest reports.42,45,46 However, adverse effects and limitations were revealed in subsequent studies, which included off-medication dyskinesia,47–49 graft-induced inflammatory responses,50 and limited tissue availability.42

    Hepatic progenitor cells represent a key target for developing new therapeutic approaches for end-stage chronic liver diseases, and isolation and transplantation of hepatic progenitor cells could represent a new therapeutic approach for liver diseases, as they offer many advantages to transplantation of mature hepatocytes.51,52 The understanding of the autocrine and paracrine pathways that regulate hepatic progenitor cell proliferation during the progression of liver diseases could open the possibility for the development of therapeutic strategies. Hepatic progenitor cells and their niche could represent, in the near future, a reserve stem cell compartment and a target for therapeutic approaches to liver disease, based on cell-specific drug delivery systems. The possibility of applying stem cell therapy to end-stage chronic liver diseases represents a critical and noteworthy goal in this field.

    5. Differentiation of iPSCs toward hepatocytes

    Orthotropic liver transplantation is the only established treatment for end-stage liver disease. However, because of the shortage of viable livers available for transplant, many patients die while still on a lengthy waiting list and many more are never added to the list. Utilization of hepatocyte transplantation and bioartificial liver devices have been proposed as alternative therapeutic approaches to this problem.53,54 The major limitation of cell therapies for liver diseases is the donor liver shortage. How to efficiently obtain a large number of liver cells is a major issue. These two approaches, however, require an unlimited source of hepatocytes, and human primary hepatocytes provide the most desirable solution for cell therapies. Yet, the utilization of primary hepatocytes in therapy has been hindered by their slow growth, loss of function, and de-differentiation in vitro.55 Because stem cells possess the ability to produce functional hepatocytes for clinical applications and drug development, such characteristics of stem cells may provide the answer to this problem. In 2006, new discoveries in the mechanisms of liver development and the emergence of iPSCs provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications.

    iPSCs are defined as reprogrammed somatic cells that have properties of pluripotent stem cells. Since the first report of the generation of iPSCs by Takahashi and Yamanaka from mouse fibroblasts in 2006,4 many studies have reported iPSC formation from species including mouse, rat, monkey, and human.56 Typically, iPSCs are generated by retroviral induction of transcription factors, Oct3/4, Sox2, KLF4, and c-Myc, in fibroblasts.56 Lentivirus and adenovirus induction, induction with other gene combinations, and virus-free approaches, such as using plasmids, small molecules, and recombinant proteins, have also been reported.56,57 This technical breakthrough in creating iPSCs from somatic cells has implications for overcoming the immunological rejection and the ethical issues associated with the derivation of ESCs from embryos. In addition, it has been shown that iPSCs can be generated from a variety of cell types, such as pancreatic cells, meningiocytes, keratinocytes, hematopoietic cells differentiated from ESCs, and primary human hepatocytes.56,58,59 iPSCs provide a potentially unlimited source for autologous cell therapy for regenerative medicine. It has been shown that human (h) iPSCs can be differentiated to many tissues, such as hematopoietic precursors and functional osteoclasts,60 pancreatic insulin-producing cells,61 cardiomyocytes,62 photoreceptors,63 as well as neural conversion.64

    Microarray analysis can present the most complete data set of this issue. Applying powerful computational techniques and the immense and growing database of known gene function to this dataset allows new insights into how similar these processes are on a global scale. To study this issue, we collected a large amount of microarray data from databases to examine the transcriptional programs of liver after partial hepatectomy, developing embryonic liver, ESCs, and iPSCs (Gene Expression Omnibus (GEO) Series (GSE), GSE10806, GSE6933, GSE6945, GSE14012, GSE10776, GSE6210, GSE10744, GSE6998).65–71 Principal component analysis (PCA) allows large datasets to be devolved into a few constructs that contain most of the variance. The results of PCA demonstrated that the time series of gene expression during liver regeneration does not segregate. Gene ontology analysis revealed that liver retrieval after hepatectomy and liver development differs dramatically, with regard to transcription factors and chromatin structure modification (Fig. 1). iPSCs are the preferred choice for hepatocyte generation because of their pluripotency and potential source for autologous hepatocyte transplantation. Differentiation of iPSCs toward a hepatic lineage has been shown in mice72,73 and in humans74–76 involving similar protocols as for hESCs. In addition, it was further shown that human iPSCs generated from foreskin fibroblasts by lentiviral transduction of OCT3/4, Sox2, Nanog, and Lin-28 homolog A (LIN28) are capable of differentiating toward functional hepatocytes by a four-step differentiation protocol and low oxygen content. Importantly, these cells proliferated in mouse fetal liver for 7 days after cell transplantation in vivo.74 Song et al reported the differentiation of human iPSCs toward functional hepatocytes with a multiphasic protocol, with 60% producing alpha-fetoprotein (AFP) and albumin, similar to hESCs differentiated to hepatocytes.75 Sullivan et al generated three human iPS lines by retroviral induction of Oct4, Sox2, Klf4, and c-Myc. They showed that all three cell lines can differentiate to hepatic endoderm, which they characterized with albumin and E-cadherin production, AFP, hepatocyte nuclear factor-4a, and cytochrome P450 7A1 expression.76 This approach allows us to evaluate gene expression during regeneration in the light of development and assess whether the dominant developmental dimensions play a significant role in regeneration. It was noted that regeneration time points did not separate significantly when transformed with developmental principal components and segregated away from development time points. From the data of the microarray analysis, it can be found that when the ESCs or iPSCs differentiate into liver progenitor cells, this will turn on the mechanism of blood vessel development, coagulation, and cell adhesion. Also, liver progenitor cells, further differentiated into liver cells, need to turn on the mechanism of oxidation reduction or metabolic process of fatty acid, carboxylic acid, etc.

    F1-1
    Fig. 1:
    The different expression gene ontology groups of liver differentiation. The microarray data from databases to check the transcription programs of liver after partial hepatectomy, developing embryonic liver, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) are shown. The results of principal component analysis (PCA) demonstrated that the time series of gene expression during liver regeneration does not segregate. Gene ontology analysis revealed that liver retrieval after hepatectomy and liver development differ dramatically with regard to transcription factors and chromatin structure modification. The results of gene ontology can be found when ESCs or iPSCs differentiate into liver progenitor cells, and turn on the mechanism of blood vessel development, coagulation, and cell adhesion. Liver progenitor cells further differentiating into liver cells is needed to turn on the mechanism of oxidation reduction or metabolic process of fatty acid and carboxylic acid.

    6. Characterization and functional evaluation of iPSC-derived hepatocytes

    There is no consensus for the characterization of hepatocyte-like cells derived from stem cells. Generally, hepatocyte-like cells are recognized by their morphology, liver-specific mRNAs, protein markers, and their functional abilities in each phase of differentiation, which are delineated by specific markers.77 Sox17, Goosecoid homeobox (GSC), and Forkhead box A2 (FOXA2) are well-known markers of definitive endoderm. Primary hepatic differentiation is often assessed by the expression of HNF3b, AFP, and transthyretin (TTR). The intermediate phase of hepatogenesis is recognized by Hepatocyte nuclear factor 1, alpha (HNF1α), Hepatocyte nuclear factor 4, alpha (HNF4α), albumin, and cytokeratin 18 (CK18). Finally, mature hepatocytes are defined by markers tryptophan oxygenase (TO), tyrosine amino-transferase (TAT), CCAAT/enhancer binding protein, alpha (C/EBPα), specific cytochrome P450 superfamily (CYPs), and asialoglycoprotein receptor 1(AGPR1).18,20,77 Several metabolic tests are used for functional assays of differentiated hepatocytes. Glycogen accumulation is examined by periodic acid shift staining as one of the mature hepatocyte characteristics.18,20,78 Hepatocyte-specific functions, such as urea synthesis and albumin production, are common functional evaluations in hepatocyte differentiation.78 Uptake of low-density lipoprotein (LDL) has also been utilized.18,20,79 Other hepatocyte-specific functions have been evaluated infrequently, such as measurement of coagulation factor VII activity80 and entry of HIV-HCV pseudotype viruses into hESC-derived hepatic cells.79

    7. Further challenges

    Although progress towards the development of improved protocols of hepatic-specific differentiation has been impressive, a number of problems exist before the cells can be used for cell transplantation trials in humans, or for toxicology or pharmacology studies. For example, xeno-free and feeder-free growth and differentiation conditions which are effective, reproducible, robust, and relatively inexpensive, must be established. This may include the development and use of small molecules and synthetic biocompatible extracellular matrix (ECMs) that can substitute for the extremely expensive growth factors and xeno-derived ECMs. The disease model with human iPSCs can offer a high throughput drug screening platform. This platform has the potential to increase the efficiency of drug development, while reducing costs and drug depletion during development.81,82

    Tumorigenicity is still one of the main obstacles in the clinical application of hiPSC and hESC, and the major question is how safe the cells must be before they can be used. Other questions, such as how does one define a lack of tumorigenicity, what is an acceptable risk, and how does one determine that a differentiated cell population is free from the presence of any early progenitor cells, must all be answered. Another major issue to be resolved is the loss of proliferation of cells when they become significantly differentiated. This leads to the question of whether a sufficient number of mature hepatocyte-like cells that have been derived from hESCs or hiPSCs can be obtained, so that cell transplantation can be clinically effective. Another question that has to be answered is at what stage cells that are undergoing differentiation should be transplanted. Should they be transplanted at an early progenitor stage when they are rapidly proliferating yet immature, or transplanted until they are fully differentiated yet much less proliferative?

    Although these are but a few of the many technical questions that must be addressed in the coming years by investigators, the characteristics of reprogrammed adult somatic cells without c-Myc, such as antioxidant properties and mobilization behavior to injury sites, may prevent oxidative stress-induced damage and provide an alternative for hepatic regeneration in acute hepatic failure (AHF), as well as an opportunity for further advances in hepatology research.

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    Keywords:

    hepatic repair; induced pluripotent stem cells

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