JRENHEP006 19..28 jrenhep.com REVIEW ARTICLE Reprogrammed Cell‐based Therapy for Liver Disease: From Lab to Clinic Amir Mehdizadeh1, Masoud Darabi2,3 1Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; 2Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; 3Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran Abstract A large number of patients are affected by liver dysfunction worldwide. Liver transplantation is the only efficient treatment in a variety of enduring liver disorders including inherent and end-stage liver diseases. The generation of human functional hepato- cytes in high quantities for liver cell therapy is an important goal for ongoing therapies in regenerative medicine. Reprogrammed cells are considered as a promising and unlimited source of hepatocytes, mainly because of their expected lack of immunogenicity and minimized ethical concerns in clinical applications. Despite gained advances in the reprogramming of somatic cells to func- tional hepatocytes in vitro, production of primary adult hepatocytes that can proliferate in vivo still remains inaccessible. As part of efforts toward translation of cell reprogramming science into clinical practice, more careful cell selection strategies should be integrated into improvement of dedifferentiation and redifferentiation protocols, especially in precision medicine where gene correction is needed. Furthermore, advances in cellular reprogramming highlight the need for developing and evaluating novel standards addressing clinical research interests in this field. Keywords: cell therapy; gene editing; liver transplantation; regenerative medicine; stem cells Received: 09 December 2016; Accepted after revision: 05 January 2017; Published: 03 February 2017. Author for correspondence: Masoud Darabi, Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz 51666-15556, Iran. Email: darabim@tbzmed.ac.ir How to cite: Mehdizadeh A et al. Reprogrammed Cell‐based Therapy for Liver Disease: From Lab to Clinic. J Ren Hepat Disord 2017;1(1):20–28. DOI: http://dx.doi.org/10.15586/jrenhep.2017.6 Copyright: Mehdizadeh A and Darabi M License: This open access article is licensed under Creative Commons Attribution 4.0 International (CC BY 4.0). http://creativecommons.org/licenses/by/4.0 Introduction A large number of patients are affected by liver dysfunction worldwide. Liver transplantation is the only efficient treat- ment in a variety of enduring liver disorders including inherent and end-stage liver diseases. However, there is a high shortage of liver organ donors causing almost 40% of patients with high rate of mortality receiving no organ transplantation. There- fore, new strategies supporting liver transplantation are in high demand. Familial hypercholesterolemia, Crigler–Najjar syndrome type I, glycogen storage disease type 1a, urea cycle defects and congenital deficiency of coagulation factor VII, hepatitis, cirrhosis, and liver cancer are the main liver dis- eases having clinical indications for cell therapy (1). There are several cell sources for human liver cell therapy, including primary hepatocytes (1), tumor cell lines (2), immor- talized hepatocyte lines from normal human hepatocytes (3), liver stem cells (4), hepatocyte-like cells from bone-marrow- derived stem cells (5), hepatocyte-like cells from fetal annex Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 mailto:darabim@tbzmed.ac.ir http://dx.doi.org/10.15586/jrenhep.2017.6 http://creativecommons.org/licenses/by/4.0 (6) and embryo (4), or reprogrammed somatic cells (7). Among them, reprogrammed cells are considered as a promising and unlimited source of hepatocytes (Figure 1), mainly because of their expected lack of immunogenicity and minimized ethi- cal concerns in clinical applications (1). These cells can be obtained by redifferentiation of any accessible somatic cells including skin, mucosa, and urine cells. In the first stage, mature somatic cells (e.g., fibroblasts) are dedifferentiated to the pluripotent stages. Besides their full pluripotency potential, these dedifferentiated cells are able to self-renew in vitro, which means they can potentially produce sufficient source for cell-based therapies. During the second stage, pluripotent cell reservoirs are induced to differentiate into functional hepatocytes. In the case of genetic deficiency, dedifferentiated cells undergo gene-editing strategies before redifferentiation. Somatic Cell Dedifferentiation The concept of somatic cell dedifferentiation into pluripotent stem cells, which are capable to form the three germinal layers and to differentiate into other cell types, provides a promising approach for regenerative medicine. This dedifferentiation technique enables us to obtain donor- or patient-specific plur- ipotent stem cells (8). In the following section, current meth- ods for dedifferentiation of somatic cells are briefly reviewed. Somatic cell nuclear transfer into oocyte The principles of this method involve in vitro removal of oocyte nucleus followed by its replacement with donor somatic nucleus. Then, cell division is stimulated by chemicals or electricity up to blastocyst stage. At this stage, cellular mass is isolated and cultured. The resulting embryonic stem (ES) cells are immunologically very identical to donor cells, and no immunosuppressant is required after transplantation to prevent their rejection. However, mitochondrial DNA from maternal oocytes could be potentially immunogenic (9). Major limitations of this method for clinical application are ethical concerns related to germ cell manipulation, chromoso- mal disorders in derived stem cells, low efficiency of transfer technique, and insufficient supply of human oocytes (8). Somatic cell fusion with embryonic stem cell An advanced method of cell fusion was developed by Cowan et al. (10) which reprogrammed human normal diploid fibro- blasts into ES cells. In this method, human embryonic cells were fused with human fibroblasts, resulting in hybrid cells with stable tetraploid DNA. Characteristics of these cells were similar to human ES cells. However, before clinical application, a set of technical limitations should be resolved. The most important challenge is to abolish ES-like cells after cell fusion. Somatic cell dedifferentiation using cell extracts Different cell extracts can alter gene expression profile in somatic cells (11). Data obtained from experiments on 293T cells, an embryonic kidney cell line, have revealed that extracts of ES cells or embryonic carcinoma cells can induce ES cell phenotype and expression of pluripotency genes. Expression of somatic gene markers such as lamin A ES cell extracts 2 1 3 4 Somatic cell Differentiation Somatic cell Somatic cell Somatic cell Nuclear transfer Unfertilized oocyte Pluripotent stem cell Mesoderm Specific differentiation Liver, kidney, lung, pancreas, stomach, intestine, bladder, germ cells Heart, muscle, blood, blood vessel, connective tissue Brain spinal cord, neurons, skin, hair, teeth, eyes, ears, nose Oct4, Sox2, Klf-4 and c-Myc gene transduction endodermendoderm Eun clea ntio n Cell fusio n Figure 1. Approaches for creating reprogrammed cells from somatic cells. 1: Somatic cell nuclear transfer into oocyte; 2, adding embryonic stem cell (ES) extract to somatic cells; 3: somatic and ES cell fusion; and 4: transduction of pluripotency genes. Gen- erated reprogrammed cells from each strategy can create three germ layers known as ectoderm, endoderm, and mesoderm. Induced redifferentiation of these reprogrammed cells can provide functional calls and tissues. Liver cell therapy using reprogrammed cells Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 21 was reduced after this manipulation. Besides, these cells gained the ability to differentiate into mesoderm and ecto- derm lineages (12). Bru et al. (13) also reported the elevation in expression of pluripotency genes including Oct3/4, Sox2, Klf-4, and c-Myc after exposure of mouse ES cell extracts to 293T cells for 48 h. However, these cells are generally limited in pluripotency potential. Somatic cell reprogramming using pluripotency-related genes In 2006, the discovery of somatic cell reprogramming to induced pluripotent stem cells (iPSCs) led to a revolution in regenerative medicine (14). iPSCs are basically patient-specific pluripotent cells that are produced by inserting four genes, including Oct4, Sox2, Kfl4, and c-Myc, necessary for fibro- blasts to evolve ES-like properties. Recent studies have indi- cated that only the presence of Oct4 gene may be sufficient to induce pluripotency in adult cells (15). In vitro, iPSCs have efficiently been used for liver tissue construction (15). Takebe et al. (16) showed that co-culture of human iPSCs- derived hepatic endoderm cells with human umbilical vein endothelial cells and human mesenchymal stem cells leads to the formation of liver buds (LBs) in 3D culture condition. Furthermore, iPSCs–LBs injection to mouse resulted in dynamic vascularization. Therefore, these cells can potentially be applied in vast areas including disease modeling, tissue engineering, and drug discovery (17, 18). Cellular Redifferentiation to Functional Liver Cells The generation of human functional hepatocytes in high quan- tities for liver cell therapy is an important goal for ongoing therapies in regenerative medicine. Here, we introduce main practical strategies for redifferentiation of pluripotent cells to liver cells. Precision medicine: CRISPR/Cas9 genome editing To date, therapies based on human ES cells are associated with controversial issues related to ethical concerns in using human embryos and potential risk of immune-mediated tis- sue rejection. Utilization of patient’s cells in order to avoid ethical concerns and rejection complications is possible by cellular reprogramming, particularly iPSCs technology (19). Based on the present protocols, fibroblasts with skin biopsy origin can be returned to pluripotent stage and serve as a renewable and autologous cellular source (20). However, the original mutation that causes disease will be present in patient-derived pluripotent stem cells. Precise correction of mutation is possible by gene-editing technique, “clustered reg- ularly interspersed short palindromic repeats (CRISPR)/Cas9 system,” which evolutionarily serves as an immune system in bacteria and archaea against virus and plasmid invasion (Figure 2). The specificity of this technique mainly depends on a guide RNA (gRNA) that can be readily reprogrammed to loci of target gene (21). Editing mutations in iPSCs derived from patients with retinitis pigmentosa was recently used Double stranded viral DNA Inactivation of viral DNA Cas/CrRNA complex Cas III Cas III Targeting viral DNA (Degradation) Creation a novel spacer Transcription Processed CrRNAs Cas IICas II Cas Cas Figure 2. Schematic presentation of clustered regularly interspersed short palindromic repeats (CRISPR/Cas9) system. It is basically a bacterial adaptive immune system. When an exogenous viral or bacteriophage genome is inserted into a bacterium, CAS protein, which acts as a nuclease, detects the exogenous unmethylated genome by attachment to a 3–5 nucleotide sequence. Then, CAS protein cuts the target sequence and inserts the fragment just before 3–5 nucleotide sequence into host genome. After transcription, crispr RNAs (crRNAs) are produced which are complementary to the exogenous genome. crRNAs can recognize the exogenous genome if a viral reinfection occurs. Mehdizadeh A and Darabi M Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 22 through CRISPR/Cas9 approach (19), which opens a promis- ing era in regenerative medicine and genome engineering. Cytokines and growth factors Hepatic regeneration is a complicated process regulated by growth factors, cytokines, transcription factors, hormones, microRNAs, metabolic pathways, and products of oxidative stress (22). The use of a specific pool of cytokines in a serum- free medium is a prerequisite for liver organogenesis step in differentiation process (23). For example, high doses of acti- vin A are widely used for endodermal induction in human pluripotent stem cells (24, 25). Some protocols have added low doses of serum for promoting essential effects of activin A in the development of endodermal induction (26, 27). Furthermore, fibroblast growth factor (FGF) and Wnt sig- naling, which play important roles in normal liver develop- ment, are also effective in endodermal induction programs (28, 29). Researchers have also combined bone morphoge- netic protein and FGFs to promote endodermal induction specificity (26, 30). Hepatocyte growth factor is also widely used in hepatic differentiation of pluripotent stem cell, mainly because of its ability in developing hepatoblast proliferation, migration, and survival through c-Met as tyrosine kinase part (31). Combination of FGF10 and retinoic acid with simultaneous inhibition of activin A is also another effective hepatic endodermal maturation protocol (32). In addition, oncostatin, which is a member of interleukin-6 family, in combination with glucocorticoids, can induce hepatocyte maturation (33, 34). Genetic and epigenetic manipulation Genetic manipulation for the purpose of overexpression of specific genes involved in hepatic induction is another approach in regenerative medicine. Transducing some tran- scription factors such as Sox17, Gata, and hepatic nuclear factor 4α elevates iPSC hepatic induction at specific time intervals in culture media (35, 36). Furthermore, epigenetic interferences have also been used to improve hepatic differen- tiation protocols (32). For instance, sodium butyrate, a speci- fic inhibitor of histone deacetylase, is frequently used to differentiate pluripotent stem cells into different cell lineages including hepatocytes in higher concentrations and longer time intervals (37–39). Chemicals (small molecules) Recent studies have proposed novel growth-factor-free proto- cols for the differentiation of pluripotent stem cells (40). Siller et al. (40) introduced a three-phasic protocol including 1) inhibition of glycogen synthase kinase 3 by CHIR99021 for definitive endoderm induction, 2) hepatic specification through dimethyl sulfoxide treatment, and 3) using dexamethasone and dihexa, a hepatocyte growth factor receptor agonist, to differentiate pluripotent stem cells into hepatocyte-like cells. Zhu et al. (41) also used a cock- tail of small molecules for incompletely reprogrammed human fibroblast cells to hepatocytes. In addition, Shan. et al. (42) identified 12,480 small molecules in a liver plat- form, and they classified them into two large groups: func- tional proliferation hits and functional hits, which were able to promote the differentiation of iPSCs and the maturation of resulted hepatocyte-like cells. Improved directed rediffer- entiation using small molecules can improve results on a cost-benefit basis in large-scale applications. Transplantation of Redifferentiated Cells in Liver Therapy The liver cell therapy procedure involves direct injection of prepared isolated cells into portal vein or spleen (43) or trans- plantation of in vitro developed tissue clusters (44). Special anatomic location of liver provides different ways for cell transplantation, including percutaneous and intravascular delivery through both portal vein and hepatic artery (45). However, studies on rat model suggest that hepatic sinusoidal delivery is the most effective approach for cell transplantation (46). Transplantation of hepatocytes under a low flow hepatic artery condition, accompanied with cellular attachment fac- tors and extracellular matrix components, is another high- throughput strategy (47). Ideally, self-regenerating capacity of transplanted liver cells is critical for cell therapy in patients with liver failure. Guo et al. (48) conditioned mice by admin- istration of retrosin, a cell cycle inhibitor, for arresting prolif- eration of native hepatocyte. After elimination of drug effects, a fresh 2 million β-galactosidase-labeled cell suspen- sion was injected into the spleen pole. Donor cell proliferation was assessed after injection of three doses of CCl4, 0.5 ml/kg. An average 20% repopulation of liver cells was recorded. More recently, post-surgery infusion of adult-derived human liver stem cells improved liver regeneration in a mouse model with 70% hepatectomy (49). Overall, the application of stem cell technology in treat- ment of liver diseases is promising at present (50). Several gene-editing clinical trials have just been approved and will be started in 2017, promoting reprogrammed cell-based therapy (51, 52). Clinical examples Inherent liver diseases A major indication for liver transplantation is inherent meta- bolic liver diseases in children (53). iPSC technology provides a unique method for designing patient- and disease-specific therapies (54). Yusa et al. (55) showed that a combination of iPSCs and a transposon-based vector technology results in biallelic correction of a point mutation in α1-antitrypsin gene which is responsible for α1-antitrypsin deficiency. Addi- tionally, genetic correction of iPSCs in patients with Wilson’s disease using a lenti-viral vector could reverse the functional Liver cell therapy using reprogrammed cells Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 23 genetic defect of Wilson’s disease gene in vitro (56). In princi- ple, genetic correction of patient-derived cells is plausible in inherent liver diseases with known mutations (Figure 3) (57). Liver failure Acute liver failure (ALF) and acute-on-chronic liver failure are two main indications for cell transplantation. iPSCs that are originated from these diseases can provide an unlimited cellular source (54). In vivo studies by Isobe et al. (58) showed that liver cells differentiated from iPSCs can save rodent from lethal drug-induced ALF. Indeed, transplanted cells exhibited proliferative and liver functional properties (59). Liver cirrhosis Because of inevitable hepatocellular damage and fibrosis of hepatic tissue in cirrhosis, therapies should mostly rely on replacement of damaged cells and fibrosis correction (54). Dif- ferent studies have reported that iPSC-derived hepatocytes promote hepatic regeneration, decrease fibrosis, and stabilize chronic liver disease in mice model (59–61). Despite these advances, iPSC-derived hepatocytes can temporarily support liver function and are hardly able to regenerate the original structure of the liver and to eliminate collagen deposition (62). Thus, other strategies are needed to help liver structure regeneration in cirrhosis through reprogramming of fibro- genic cells or transplantation of liver tissue construct (62). Liver cancer It has been reported that downregulation of cyclin-dependent kinase inhibitor 1, an important cell cycle mediator, in pluri- potent stem cells generated from patients with hepatocellular carcinoma can promote differentiation into normal human hepatoma-like cells (63). Furthermore, it was shown that inhibition of aldo-ketoreductase 1 member B10 promotes retinoic acid-induced differentiation. However, efficacy and patient specificity of the first-mentioned method seem to be higher, as it avoids the toxic effects of combination therapy (63). Lei et al. (64) also introduced a protocol for generating cytotoxic T lymphocytes from iPSCs as an unlimited cellular source in breast cancer therapy. In future, this strategy can be used as a novel method for liver cancer. Clinical limitations Using viral vectors for transducing Oct4, Sox2, Klf4, and c- Myc is an exciting method for generating human iPSCs. Despite high efficacy of this procedure, there remain some critical limitations for the application of iPSCs in clinics (65). Applications of retroviral-generated iPSCs are limited because of the 1) integration of retroviral DNA into host gen- ome with variable copy numbers which interfere with promo- ter elements, polyadenylation signals, and coding sequences, affecting transcription potency (20), and 2) loss of pluripo- tency potential because of low expression of exogenous Patient with inherent liver disease Mutation correction using CRISPR/CAS9 technology Healthy iPSCsDiseased iPSCsPatient derived skin Fibroblasts Differentiated healthy cells Cell transplantation (Functional, efficient, safe) Figure 3. Clinical application of clustered regularly interspersed short palindromic repeats (CRISPR/Cas9). Skin fibroblasts from patient source can be dedifferentiated to pluripotent stem cells containing the disease causing mutation. This mutation can be corrected by CRISPR/Cas9 technology resulting in healthy stem cells which can be redifferentiated to patient-specific healthy cells for transplantation. Mehdizadeh A and Darabi M Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 24 Oct4, Sox2, Klf4, and c-Myc from viral constructs (66). Another critical concern is the exogenous genes itself; overex- pression of Oct4, Sox2, Klf4, and c-Myc increases the chance of tumorigenesis (66, 67). Overexpression of Oct4 induces epithelial cell dysplasia (68). Sox2 overexpression is also asso- ciated with serrated adenoma and mucinous colon carcinoma (69). Klf4 and c-Myc are also associated with breast cancer and some other human carcinomas (70). Tumor progression is also observed in murine chimeras after injection of retro- viral-generated iPSCs to blastocysts which has been attribu- ted to c-Myc overexpression (71, 72). Despite gained advances in the differentiation of pluripo- tent stem cells into functional hepatocytes in vitro, produc- tion of primary adult hepatocytes that can proliferate in vivo still remains inaccessible (73, 74). The reason for this pro- blem is that expansion and proliferation of transplanted cells need a tense sustain of hepatic cell mass (75). Furthermore, besides pluripotent stem cells-related problems, cell delivery complications are another important limiting factor of liver cell therapy. Direct injection of cells to liver parenchyma may increase the risk of cell entry to hepatic vein outflow and pulmonary vein, causing embolic complications (76). Injection of cells to hepatic or splenic artery, theoretically, also seems to be achievable. However, these methods may increase the risk of tissue necrosis due to embolic occlusion of vessels. In high blood flow condition, engrafted cells may be destroyed because of incoming mechanical forces (47). Furthermore, in portal hypertension and chronic liver dis- eases, the transplanted cell may be translocated to lungs through portosystemic collaterals or channels causing cardio- vascular problems (Figure 4) (45,77). Therefore, there is an urgent need for clinical trial designing for the application of successful cell delivery methods to liver sinusoids. Obviously, standards of professional practice play an important role in the clinical setting. There is no international standard for reprogrammed liver cell therapy, as cell therapy in general has been limited to heterologous primary cells with resource scarcity and little satisfactory outcome. The major obstacle in hepatocyte transplantation is poor engraftment results, encouraging researchers to suggest new strategies. Prominent among these are modifying metabolic status in the recipients of liver cell therapy (78) and co- transplantation of mesenchymal stem cells (79) because of their significant effects on liver regeneration and repair. Conclusion The restoration of hepatic function by patient-specific cell transplantation remains a promising strategy for liver ther- apy. Reprogramming strategy exploits preexisting somatic cells to produce other mature cell types or progenitors. The cornerstone of this strategy is to keep the cellular genome sta- bility during dedifferentiation and efficient redifferentiation. Patient-derived hepatic cells can be transplanted directly in the form of isolated cells or as in vitro-generated liver tissue constructs. Animal model data suggest that liver tissue con- structs may offer better regeneration and improved survival, but teratoma formation and rejection by immune system are observed in both strategies. These occur primarily due to the presence of residual undifferentiated cells in hepato- cytes derived from human iPSCs. As a part of efforts toward translation of cell reprogramming science into clinical prac- tice, more careful cell selection strategies should be integrated into improvement of dedifferentiation and redifferentiation protocols, especially in precision medicine where gene correc- tion is needed. Furthermore, advances in cellular reprogram- ming highlight the need for developing and evaluating novel standards addressing clinical research interests in this field. Acknowledgments The research was financially supported by grants from the Stem Cell Research Center at Tabriz University of Medical Sciences and Iranian Council for Development of Stem Cell Sciences and Technologies. The authors would like to thank the Liver and Gastrointestinal Diseases Research Center at Intraportal infusion Catheter through umbilical vein (in newborn) Hickman line in inferior messenteric vein Figure 4. Liver cell transplantation routs. The portal circulation can pass directly infused cells to the liver tissue. The routine ways to directly access the portal circulation are percutaneous intraportal infusion, umbilical catheterization, and insertion of Hickman line in inferior mesenteric vein. The main concerns for transplantation of reprogrammed cells include using fresh or cryopreserved hepatocytes with cell viability of more than 60%, a minimum of 109 cell/infusion, and portal pressure monitoring (77). Liver cell therapy using reprogrammed cells Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 25 Tabriz University of Medical Sciences for support of this work. The authors express acknowledgements to their many colleagues whose related contribution was not cited here. Conflict of interest The authors declare no conflicts of interest with respect to research, authorship, and/or publication of this article. References 1. Yu Y, Fisher JE, Lillegard JB, Rodysill B, Amiot B, Nyberg SL. Cell therapies for liver diseases. Liver Transpl. 2012;18(1):9–21. http://dx.doi.org/10.1002/lt.22467 2. Ellis AJ, Hughes RD, Wendon JA, Dunne J, Langley PG, Kelly JH, et al. Pilot‐controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology. 1996;24(6): 1446–51. http://dx.doi.org/10.1002/hep.510240625 3. Chen Y, Li J, Liu X, Zhao W, Wang Y, Wang X. Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice. Transplant Proc. 2010; 42: 1907–14. http://dx.doi.org/10.1016/j.transproceed.2010.01.061 4. Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas. Science. 2008;322(5907):1490–4. http:// dx.doi.org/10.1126/science.1161431 5. Li H, Zhang B, Lu Y, Jorgensen M, Petersen B, Song S. Adipose tissue-derived mesenchymal stem cell-based liver gene delivery. J Hepatol. 2011;54(5):930–8. http://dx.doi.org/10.1016/j.jhep. 2010.07.051 6. Piscaglia AC, Campanale M, Gasbarrini A, Gasbarrini G. Stem cell-based therapies for liver diseases: State of the art and new perspectives. Stem Cells Int. 2010;2010:259461. http://dx.doi. org/10.4061/2010/259461 7. Si‐Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, et al. Highly efficient generation of human hepatocyte–like cells from induced pluripotent stem cells. Hepatology. 2010;51 (1):297–305. http://dx.doi.org/10.1002/hep.23354 8. Ramesh T, Lee S-H, Lee C-S, Kwon Y-W, Cho H-J. Somatic cell dedifferentiation/reprogramming for regenerative medicine. Int J Stem Cells. 2009;2(1):18. http://dx.doi.org/10.15283/ijsc.2009.2. 1.18 9. Brambrink T, Hochedlinger K, Bell G, Jaenisch R. ES cells derived from cloned and fertilized blastocysts are transcriptionally and func- tionally indistinguishable. Proc Natl Acad Sci U S A. 2006;103(4): 933–8. http://dx.doi.org/10.1073/pnas.0510485103 10. Cowan CA, Atienza J, Melton DA, Eggan K. Nuclear repro- gramming of somatic cells after fusion with human embryonic stem cells. Science. 2005;309(5739):1369–73. http://dx.doi.org/ 10.1126/science.1116447 11. Håkelien A-M, Landsverk HB, Robl JM, Skålhegg BS, Collas P. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat Biotechnol. 2002;20(5):460–6. http://dx.doi.org/10. 1038/nbt0502-460 12. Taranger CK, Noer A, Sørensen AL, Håkelien A-M, Boquest AC, Collas P. Induction of dedifferentiation, genome- wide transcriptional programming, and epigenetic reprogram- ming by extracts of carcinoma and embryonic stem cells. Mol Biol Cell. 2005;16(12):5719–35. http://dx.doi.org/10.1091/mbc. E05-06-0572 13. Bru T, Clarke C, McGrew MJ, Sang HM, Wilmut I, Blow JJ. Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts. Exp Cell Res. 2008;314 (14):2634–42. http://dx.doi.org/10.1016/j.yexcr.2008.05.009 14. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76. http://dx.doi.org/10. 1016/j.cell.2006.07.024 15. Kim JB, Greber B, Araúzo-Bravo MJ, Meyer J, Park KI, Zaehres H, et al. Direct reprogramming of human neural stem cells by OCT4. Nature. 2009;461(7264):649–3. http://dx.doi. org/10.1038/nature08436. PubMed PMID: 19718018. 16. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 2013;499(7459): 481–4. http://dx.doi.org/10.1038/nature12271 17. Hansel MC, Davila JC, Vosough M, Gramignoli R, Skvorak KJ, Dorko K, et al. The use of induced pluripotent stem cells for the study and treatment of liver diseases. Curr Protoc Toxicol. 2016;67:14.3.1–13.27. http://dx.doi.org/10.1002/0471140856. tx1413s67. PubMed PMID: 26828329; PubMed Central PMCID: PMCPMC4795152. 18. Goldman O, Gouon-Evans V. Human pluripotent stem cells: Myths and future realities for liver cell therapy. Cell Stem Cell. 2016;18(6):703–6. http://dx.doi.org/10.1016/j.stem.2016.05.019. PubMed PMID: 27257759. 19. Bassuk AG, Zheng A, Li Y, Tsang SH, Mahajan VB. Precision medicine: Genetic repair of retinitis pigmentosa in patient- derived stem cells. Sci Rep. 2016;6:19969. http://dx.doi.org/10. 1038/srep19969 20. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5): 861–72. http://dx.doi.org/10.1016/j.cell.2007.11.019 21. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endo- nuclease in adaptive bacterial immunity. Science. 2012;337 (6096):816–21. http://dx.doi.org/10.1126/science.1225829 22. Hu C, Li L. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration. Protein Cell. 2015;6(8):562–74. http://dx.doi.org/10.1007/s13238- 015-0180-2 23. Han S, Bourdon A, Hamou W, Dziedzic N, Goldman O, Gouon-Evans V. Generation of functional hepatic cells from pluripotent stem cells. J Stem Cell Res Ther. 2012;10(8):1–7. http://dx.doi.org/10.4172/2157-7633.s10-008 24. Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, et al. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology. 2007;45(5):1229–39. http://dx.doi. org/10.1002/hep.21582 25. Liu H, Ye Z, Kim Y, Sharkis S, Jang YY. Generation of endo- derm‐derived human induced pluripotent stem cells from pri- mary hepatocytes. Hepatology. 2010;51(5):1810–9. http://dx. doi.org/10.1002/hep.23626 26. Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells. 2008;26(5):1117–27. http://dx.doi.org/10.1634/stem cells.2007-1102 27. Liu H, Kim Y, Sharkis S, Marchionni L, Jang Y-Y. In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins. Sci Transl Med. 2011;3(82):82ra39. http:// dx.doi.org/10.1126/scitranslmed.3002376 28. Hay DC, Fletcher J, Payne C, Terrace JD, Gallagher RC, Snoeys J, et al. Highly efficient differentiation of hESCs to func- tional hepatic endoderm requires ActivinA and Wnt3a signaling. Mehdizadeh A and Darabi M Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 26 http://dx.doi.org/10.1002/lt.22467 http://dx.doi.org/10.1002/hep.510240625 http://dx.doi.org/10.1016/j.transproceed.2010.01.061 http://dx.doi.org/10.1126/science.1161431 http://dx.doi.org/10.1126/science.1161431 http://dx.doi.org/10.1016/j.jhep.2010.07.051 http://dx.doi.org/10.1016/j.jhep.2010.07.051 http://dx.doi.org/10.4061/2010/259461 http://dx.doi.org/10.4061/2010/259461 http://dx.doi.org/10.1002/hep.23354 http://dx.doi.org/10.15283/ijsc.2009.2.1.18 http://dx.doi.org/10.15283/ijsc.2009.2.1.18 http://dx.doi.org/10.1073/pnas.0510485103 http://dx.doi.org/10.1126/science.1116447 http://dx.doi.org/10.1126/science.1116447 http://dx.doi.org/10.1038/nbt0502-460 http://dx.doi.org/10.1038/nbt0502-460 http://dx.doi.org/10.1091/mbc.E05-06-0572 http://dx.doi.org/10.1091/mbc.E05-06-0572 http://dx.doi.org/10.1016/j.yexcr.2008.05.009 http://dx.doi.org/10.1016/j.cell.2006.07.024 http://dx.doi.org/10.1016/j.cell.2006.07.024 http://dx.doi.org/10.1038/nature08436 http://dx.doi.org/10.1038/nature08436 http://dx.doi.org/10.1038/nature12271 http://dx.doi.org/10.1002/0471140856.tx1413s67 http://dx.doi.org/10.1002/0471140856.tx1413s67 http://dx.doi.org/10.1016/j.stem.2016.05.019 http://dx.doi.org/10.1038/srep19969 http://dx.doi.org/10.1038/srep19969 http://dx.doi.org/10.1016/j.cell.2007.11.019 http://dx.doi.org/10.1126/science.1225829 http://dx.doi.org/10.1007/s13238-015-0180-2 http://dx.doi.org/10.1007/s13238-015-0180-2 http://dx.doi.org/10.4172/2157-7633.s10-008 http://dx.doi.org/10.1002/hep.21582 http://dx.doi.org/10.1002/hep.21582 http://dx.doi.org/10.1002/hep.23626 http://dx.doi.org/10.1002/hep.23626 http://dx.doi.org/10.1634/stemcells.2007-1102 http://dx.doi.org/10.1634/stemcells.2007-1102 http://dx.doi.org/10.1126/scitranslmed.3002376 http://dx.doi.org/10.1126/scitranslmed.3002376 Proc Natl Acad Sci U S A. 2008;105(34):12301–6. http://dx.doi. org/10.1073/pnas.0806522105 29. Morrison GM, Oikonomopoulou I, Migueles RP, Soneji S, Livigni A, Enver T, et al. Anterior definitive endoderm from ESCs reveals a role for FGF signaling. Cell Stem Cell. 2008; 3(4):402–15. http://dx.doi.org/10.1016/j.stem.2008.07.021 30. Gouon-Evans V, Boussemart L, Gadue P, Nierhoff D, Koehler CI, Kubo A, et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm. Nat Biotechnol. 2006;24(11):1402–11. http://dx.doi.org/10. 1038/nbt1258 31. Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschiesche W, Sharpe M, et al. Scatter factor/hepatocyte growth factor is essen- tial for liver development. Nature. 1995;373(6516):699–702. http://dx.doi.org/10.1038/373699a0 32. Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, et al. Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology. 2010;51(5): 1754–65. http://dx.doi.org/10.1002/hep.23506 33. Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J. 1999;18(8):2127–36. http://dx.doi.org/10.1093/emboj/18.8.2127 34. Rahimi Y, Mehdizadeh A, Nozad Charoudeh H, Nouri M, Valaei K, Fayezi S, et al. Hepatocyte differentiation of human induced pluripotent stem cells is modulated by stearoyl-CoA desaturase 1 activity. Dev Growth Differ. 2015;57(9):667–74. http://dx.doi.org/10.1111/dgd.12255. PubMed PMID: 26676854. 35. Takayama K, Inamura M, Kawabata K, Katayama K, Higuchi M, Tashiro K, et al. Efficient generation of functional hepato- cytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction. Mol Ther. 2012;20(1):127– 37. http://dx.doi.org/10.1038/mt.2011.234 36. Takayama K, Inamura M, Kawabata K, Tashiro K, Katayama K, Sakurai F, et al. Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction. PLoS One. 2011;6(7):e21780. http://dx.doi.org/10.1371/journal. pone.0021780 37. Kretsovali A, Hadjimichael C, Charmpilas N. Histone deacety- lase inhibitors in cell pluripotency, differentiation, and repro- gramming. Stem Cells Int. 2012;2012:184154. http://dx.doi.org/ 10.1155/2012/184154 38. Ren M, Yan L, Shang CZ, Cao J, Lu LH, Min J, et al. Effects of sodium butyrate on the differentiation of pancreatic and hepatic progenitor cells from mouse embryonic stem cells. J Cell Biochem. 2010;109(1):236–44. 39. Zhou QJ, Xiang LX, Shao JZ, Hu RZ, Lu YL, Yao H, et al. In vitro differentiation of hepatic progenitor cells from mouse embryonic stem cells induced by sodium butyrate. J Cellular Biochem. 2007;100(1):29–42. http://dx.doi.org/10.1002/ jcb.20970 40. Siller R, Greenhough S, Naumovska E, Sullivan GJ. Small- molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. 2015;4(5):939–52. http://dx.doi. org/10.1016/j.stemcr.2015.04.001 41. Zhu S, Rezvani M, Harbell J, Mattis AN, Wolfe AR, Benet LZ, et al. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature. 2014;508(7494):93–7. http://dx.doi. org/10.1038/nature13020 42. Shan J, Schwartz RE, Ross NT, Logan DJ, Thomas D, Duncan SA, et al. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol. 2013;9(8): 514–20. http://dx.doi.org/10.1038/nchembio.1270 43. Wertheim JA, Baptista PM, Soto-Gutierrez A. Cellular therapy and bioartificial approaches to liver replacement. Curr Opin Organ Transplant. 2012;17(3):235. http://dx.doi.org/10.1097/ MOT.0b013e3283534ec9 44. Bao J, Shi Y, Sun H, Yin X, Yang R, Li L, et al. Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats. Cell Transplant. 2011;20(5):753–66. http://dx.doi.org/10.3727/ 096368910X536572. PubMed PMID: 21054928. 45. Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol. 2015; 62(1):S157–69. http://dx.doi.org/10.1016/j.jhep.2015.02.040 46. Gupta S, Rajvanshi P, Sokhi R, Slehria S, Yam A, Kerr A, et al. Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology. 1999;29(2):509–19. http://dx.doi.org/10.1002/hep. 510290213 47. Gupta S, Lee CD, Vemuru RP, Bhargava KK. 111Indium label- ing of hepatocytes for analysis of short‐term biodistribution of transplanted cells. Hepatology. 1994;19(3):750–7. http://dx.doi. org/10.1002/hep.510290213 48. Guo D, Fu T, Nelson JA, Superina RA, Soriano HE. Liver repopulation after cell transplantation in mice treated with retro- rsine and carbon tetrachloride1. Transplantation. 2002;73(11): 1818–24. http://dx.doi.org/10.1097/00007890-200206150-00020 49. Herrero A, Prigent J, Lombard C, Rosseels V, Daujat- Chavanieu M, Breckpot K, et al. Adult-derived human liver stem/progenitor cells infused 3 days post-surgery improve liver regeneration in a mouse model of extended hepatectomy. Cell Transplant. 2016; ahead of print. http://dx.doi.org/10.3727/ 096368916X692960. PubMed PMID: 27657746. 50. Trounson A, DeWitt ND. Pluripotent stem cells progressing to the clinic. Nat Rev Mol Cell Biol. 2016;17(3):194–200. http:// dx.doi.org/10.1038/nrm.2016.10 51. Cyranoski D. Chinese scientists to pioneer first human CRISPR trial. Nature. 2016;535(7613):476–7. http://dx.doi.org/10.1038/ nature.2016.20302. PubMed PMID: 27466105. 52. Deng P, Torrest A, Pollock K, Dahlenburg H, Annett G, Nolta JA, et al. Clinical trial perspective for adult and juvenile Huntington’s disease using genetically-engineered mesenchymal stem cells. Neural Regen Res. 2016;11(5):702–5. http://dx.doi. org/10.4103/1673-5374.182682. PubMed PMID: 27335539; PubMed Central PMCID: PMCPMC4904446. 53. Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, et al. Treatment of the Crig- ler–Najjar syndrome type I with hepatocyte transplantation. N Engl J Med. 1998;338(20):1422–7. http://dx.doi.org/10.1056/ NEJM199805143382004 54. Yu Y, Wang X, Nyberg SL. Application of induced pluripotent stem cells in liver diseases. Cell Med. 2014;7(1):1–13. http://dx. doi.org/10.3727/215517914X680056 55. Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011;478 (7369):391–4. http://dx.doi.org/10.1038/nature10424 56. Zhang S, Chen S, Li W, Guo X, Zhao P, Xu J, et al. Rescue of ATP7B function in hepatocyte-like cells from Wilson’s disease induced pluripotent stem cells using gene therapy or the chaper- one drug curcumin. Hum Mol Genet. 2011;20(16):3176–87. http://dx.doi.org/10.1093/hmg/ddr223 Liver cell therapy using reprogrammed cells Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 27 http://dx.doi.org/10.1073/pnas.0806522105 http://dx.doi.org/10.1073/pnas.0806522105 http://dx.doi.org/10.1016/j.stem.2008.07.021 http://dx.doi.org/10.1038/nbt1258 http://dx.doi.org/10.1038/nbt1258 http://dx.doi.org/10.1038/373699a0 http://dx.doi.org/10.1002/hep.23506 http://dx.doi.org/10.1093/emboj/18.8.2127 http://dx.doi.org/10.1111/dgd.12255 http://dx.doi.org/10.1038/mt.2011.234 http://dx.doi.org/10.1371/journal.pone.0021780 http://dx.doi.org/10.1371/journal.pone.0021780 http://dx.doi.org/10.1155/2012/184154 http://dx.doi.org/10.1155/2012/184154 http://dx.doi.org/10.1002/jcb.20970 http://dx.doi.org/10.1002/jcb.20970 http://dx.doi.org/10.1016/j.stemcr.2015.04.001 http://dx.doi.org/10.1016/j.stemcr.2015.04.001 http://dx.doi.org/10.1038/nature13020 http://dx.doi.org/10.1038/nature13020 http://dx.doi.org/10.1038/nchembio.1270 http://dx.doi.org/10.1097/MOT.0b013e3283534ec9 http://dx.doi.org/10.1097/MOT.0b013e3283534ec9 http://dx.doi.org/10.3727/096368910X536572 http://dx.doi.org/10.3727/096368910X536572 http://dx.doi.org/10.1016/j.jhep.2015.02.040 http://dx.doi.org/10.1002/hep.510290213 http://dx.doi.org/10.1002/hep.510290213 http://dx.doi.org/10.1002/hep.510290213 http://dx.doi.org/10.1002/hep.510290213 http://dx.doi.org/10.1097/00007890-200206150-00020 http://dx.doi.org/10.3727/096368916X692960 http://dx.doi.org/10.3727/096368916X692960 http://dx.doi.org/10.1038/nrm.2016.10 http://dx.doi.org/10.1038/nrm.2016.10 http://dx.doi.org/10.1038/nature.2016.20302 http://dx.doi.org/10.1038/nature.2016.20302 http://dx.doi.org/10.4103/1673-5374.182682 http://dx.doi.org/10.4103/1673-5374.182682 http://dx.doi.org/10.1056/NEJM199805143382004 http://dx.doi.org/10.1056/NEJM199805143382004 http://dx.doi.org/10.3727/215517914X680056 http://dx.doi.org/10.3727/215517914X680056 http://dx.doi.org/10.1038/nature10424 http://dx.doi.org/10.1093/hmg/ddr223 57. Chun YS, Chaudhari P, Jang Y-Y. Applications of patient-spe- cific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease. Int J Biol Sci. 2010;6(7):796–805. http://dx.doi.org/10.7150/ijbs.6.796 58. Isobe K, Cheng Z, Ito S, Nishio N. Aging in the mouse and per- spectives of rejuvenation through induced pluripotent stem cells (iPSCs). Results Probl Cell Differ. 2012;55:413–27. http://dx.doi. org/10.1007/978-3-642-30406-4_21 59. Espejel S, Roll GR, McLaughlin KJ, Lee AY, Zhang JY, Laird DJ, et al. Induced pluripotent stem cell–derived hepato- cytes have the functional and proliferative capabilities needed for liver regeneration in mice. J Clin Invest. 2010;120(9): 3120–6. http://dx.doi.org/10.1172/JCI43267 60. Asgari S, Moslem M, Bagheri-Lankarani K, Pournasr B, Miryounesi M, Baharvand H. Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells. Stem Cell Rev Rep. 2013;9(4):493–504. http://dx.doi.org/10. 1007/s12015-011-9330-y 61. Choi SM, Kim Y, Liu H, Chaudhari P, Ye Z, Jang Y-Y. Liver engraftment potential of hepatic cells derived from patient-speci- fic induced pluripotent stem cells. Cell Cycle. 2011;10(15): 2423–7. http://dx.doi.org/10.4161/cc.10.15.16869 62. Chen Z, Qi L, Zeng R, Li HY, Dai LJ. Stem cells and hepatic cirrhosis. Panminerva Med. 2010;52(2):149–65. 63. Moriguchi H, Chung RT, Sato C. An identification of novel ther- apy for human hepatocellular carcinoma by using human induced pluripotent stem cells. Hepatology. 2010;51(3):1090–1. 64. Lei F, Zhao B, Haque R, Xiong X, Budgeon L, Christensen ND, et al. In vivo programming of tumor antigen-specific T lympho- cytes from pluripotent stem cells to promote cancer immunosur- veillance. Cancer Res. 2011;71(14):4742–7. http://dx.doi.org/10. 1158/0008-5472.CAN-11-0359 65. Medvedev SP, Shevchenko AI, Zakian SM. Induced pluripotent stem cells: Problems and advantages when applying them in regenerative medicine. Acta Naturae. 2010;2(2):18–28. 66. Okita K, Ichisaka T, Yamanaka S. Generation of germline-com- petent induced pluripotent stem cells. Nature. 2007;448(7151): 313–7. http://dx.doi.org/10.1038/nature05934 67. Carey BW, Markoulaki S, Hanna J, Saha K, Gao Q, Mitalipova M, et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A. 2009;106(1):157–62. http://dx.doi.org/10.1073/ pnas.0811426106 68. Hochedlinger K, Yamada Y, Beard C, Jaenisch R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell. 2005;121(3):465–77. http://dx.doi.org/10.1016/j.cell.2005.02.018 69. Park ET, Gum JR, Kakar S, Kwon SW, Deng G, Kim YS. Aberrant expression of SOX2 upregulates MUC5AC gastric foveolar mucin in mucinous cancers of the colorectum and related lesions. Int J Cancer. 2008;122(6):1253–60. http://dx. doi.org/10.1002/ijc.23225 70. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. The diverse functions of Krüppel‐like factors 4 and 5 in epithelial biology and pathobiology. Bioessays. 2007;29(6):549–57. http:// dx.doi.org/10.1002/bies.20581 71. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 2008;322(5903):949–53. http://dx.doi.org/ 10.1126/science.1164270 72. Duinsbergen D, Salvatori D, Eriksson M, Mikkers H. Tumors originating from induced pluripotent stem cells and methods for their prevention. Ann N Y Acad Sci. 2009;1176(1): 197–204. http://dx.doi.org/10.1111/j.1749-6632.2009.04563.x 73. Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. 2010;120(9):3127–36. http://dx.doi.org/10.1172/JCI43122 74. Hashemi Goradel N, Darabi M, Shamsasenjan K, Ejtehadifar M, Zahedi S. Methods of liver stem cell therapy in rodents as models of human liver regeneration in hepatic failure. Adv Pharm Bull. 2015;5(3):293–8. http://dx.doi.org/10.5681/apb.2015.041. PubMed PMID: 26504749; PubMed Central PMCID: PMCPMC4616895. 75. Puppi J, Strom SC, Hughes RD, Bansal S, Castell JV, Dagher I, et al. Improving the techniques for human hepatocyte trans- plantation: Report from a consensus meeting in London. Cell Transplant. 2012;21(1):1–10. http://dx.doi.org/10.3727/ 096368911X566208 76. Nagata H, Ito M, Shirota C, Edge A, McCowan TC, Fox IJ. Route of hepatocyte delivery affects hepatocyte engraftment in the spleen1. Transplantation. 2003;76(4):732–4. http://dx.doi. org/10.1097/01.TP.0000081560.16039.67 77. Kirk AD, Knechtle SJ, Larsen CP, Madsen JC, Pearson TC, Webber SA. Textbook of organ transplantation set. Hoboken, NJ: John Wiley & Sons; 2014. 78. Hashemi Goradel N, Eghbal MA, Darabi M, Roshangar L, Asadi M, Zarghami N, et al. Improvement of liver cell therapy in rats by dietary stearic acid. Iran Biomed J. 2016;20(4): 217–22.PubMed PMID: 27090202; PubMed Central PMCID: PMCPMC4983676. 79. Liu T, Wang Y, Tai G, Zhang S. Could co‐transplantation of iPS cells derived hepatocytes and MSCs cure end‐stage liver dis- ease? Cell Biol Int. 2009;33(11):1180–3. http://dx.doi.org/10. 1016/j.cellbi.2009.08.007 Mehdizadeh A and Darabi M Codon Publications Journal of Renal and Hepatic Disorders 2017; 1(1): 20–28 28 http://dx.doi.org/10.7150/ijbs.6.796 http://dx.doi.org/10.1007/978-3-642-30406-4_21 http://dx.doi.org/10.1007/978-3-642-30406-4_21 http://dx.doi.org/10.1172/JCI43267 http://dx.doi.org/10.1007/s12015-011-9330-y http://dx.doi.org/10.1007/s12015-011-9330-y http://dx.doi.org/10.4161/cc.10.15.16869 http://dx.doi.org/10.1158/0008-5472.CAN-11-0359 http://dx.doi.org/10.1158/0008-5472.CAN-11-0359 http://dx.doi.org/10.1038/nature05934 http://dx.doi.org/10.1073/pnas.0811426106 http://dx.doi.org/10.1073/pnas.0811426106 http://dx.doi.org/10.1016/j.cell.2005.02.018 http://dx.doi.org/10.1002/ijc.23225 http://dx.doi.org/10.1002/ijc.23225 http://dx.doi.org/10.1002/bies.20581 http://dx.doi.org/10.1002/bies.20581 http://dx.doi.org/10.1126/science.1164270 http://dx.doi.org/10.1126/science.1164270 http://dx.doi.org/10.1111/j.1749-6632.2009.04563.x http://dx.doi.org/10.1172/JCI43122 http://dx.doi.org/10.5681/apb.2015.041 http://dx.doi.org/10.3727/096368911X566208 http://dx.doi.org/10.3727/096368911X566208 http://dx.doi.org/10.1097/01.TP.0000081560.16039.67 http://dx.doi.org/10.1097/01.TP.0000081560.16039.67 http://dx.doi.org/10.1016/j.cellbi.2009.08.007 http://dx.doi.org/10.1016/j.cellbi.2009.08.007