Academia Journal of Biotechnology 4(8): 297-306, August 2016 DOI: 10.15413/ajb.2016.0112 ISSN: 2315-7747 ©2016 Academia Publishing

Research Paper

Induced Pluripotent Stem Cells Generated from Patients with Chronic Liver Failure

Accepted 19th May, 2016 ABSTRACT Human induced pluripotent stem cells (iPSCs) technology offers opportunities to reprogram adult cells into embryonic-like stem cells (ESCs), enabling the development of novel stem cell-based therapeutic approaches for regenerative medicine. The objective of this study was to generate and characterize human iPSCs from adult cells of a patient with liver failure, which might provide a potential new source of cells for hepatocyte transplantation. Human iPSCs were induced from dermal fibroblasts of a 63 year-old adult patient with chronic liver failure by introducing four factors: OCT4, SOX2, C-MYC and KLF4 under ES cell culture conditions. The expressions of ESCs marker genes and proteins were monitored by RT-PCR and immunofluorescence, respectively. The pluripotency of iPSCs was determined by in vivo teratoma formation and in vitro embryoid bodies differentiation analyses. These cells exhibited the morphology and growth properties of ESCs. All iPSCs examined expressed ESC markers, such as OCT4, SOX2, FGF4, KLF4, ERAS, DAX1, ESG1, SSEA-4, TRA-1-60, TRA-1-81 and NANOG. Additionally, all human lines maintained a normal 46 XY karyotype. Subcutaneous transplantation of iPSCs into immune-deficient mice resulted in tumor formation. The tumors contained a variety of tissues from all three germ layers. The embryoid bodies were allowed to spontaneously differentiate. Our data demonstrated that iPSCs can be successfully generated from a dermal fibroblast of an adult patient with chronic liver failure, which may provide an inexhaustible cell source for hepatocyte transplantation to patients with end-stage liver disease. Key words: Embryonic stem cells, dermal fibroblasts, induced pluripotent stem cells, liver failure.

INTRODUCTION Chronic liver failure is a frequent, life-threatening condition in patients with end-stage liver disease. Once a patient develops hepatic de-compensation, liver transplantation is the definitive treatment for those qualified (Arora and Keeffe, 2008). Although, this procedure is effective in the reduction of mortality, less than one third of patients are able to obtain a donor liver.

Hepatocyte transplantation has been demonstrated to restore damaged liver function in host animals and serves as a promising alternative for the treatment of a wide range of liver diseases (Enns and Millan, 2008).

Embryonic stem cells (ESCs) isolated from the inner cell mass of mammalian blastocysts represent an immortal propagation of pluripotent cells that can theoretically differentiate into any tissue cells, including hepatocytes. Thus, ESCs can serve as an inexhaustible cell source for hepatocyte transplantation. However, there are ethical difficulties regarding the use of human embryos, as well as, the problem of tissue rejection following transplantation in patients. One way to circumvent these issues is the generation of pluripotent cells directly from the patients’ own somatic cells.

Hai Lu1,4,, Jinqun Jiang2, Zhan Huang1, Qingsong Wu3 and Yi Gao4* 1Department of Surgical Oncology, The Yuebei Hospital of Guangdong Province; Shaoguan 512026; China. 2Department of Clinical Laboratory, The Yuebei Hospital of Guangdong Province; Shaoguan 512026; China. 3Department of Hepatobiliary Surgery; The Yuebei Hospital of Guangdong Province; Shaoguan 512026; China. 4Department of Hepatobiliary Surgery (Division Ⅱ), Zhujiang Hospital; Southern Medical University; Guangzhou 510262; China. *Corresponding author. E-mail: 927988949@qq.com Tel.: +86-0751-6913231.

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Recently, induced pluripotent stem cells (iPSCs) have been reprogrammed directly from fibroblast, keratinocytes or blood cells by introducing defined transcription factors such as OCT4, SOX2, KLF4, C-MYC and Utf1 (Takahashi et al., 2007; Staerk et al., 2010; Zhao et al., 2008; Ebert et al., 2008). The resulting iPSCs share similar functional and molecular phenotypic characteristics with ES cells. This opens new opportunities for regenerative medicine and in vitro disease modeling.

Human iPSCs have since been generated from patients with various diseases, including spinal muscular atrophy (Ebert et al., 2008), myotrophic lateral sclerosis (Dimos et al., 2008), dyskeratosis congenita patients inherited from metabolic disorders of the liver (Rashid ST,et al,2010), schizophrenia (Chiang et al., 2011), Hurler syndrome (Tolar et al., 2011), Down syndrome (Mou et al., 2012), type 1 diabetes (Kudva et al., 2012; Maehr et al., 2009) and heart failure (Zwi-Dantsis et al., 2012), but not liver failure.

Herein, the generation and characterization of human iPSCs lines by introducing OCT4, SOX2, KLF4 and C-MYC factors into dermal fibroblasts from a patient with chronic liver failure was reported. Our human iPSCs are indistinguishable from hESCs with respect to colony morphology, surface and pluripotency markers, normal karyotype, and differentiation potential. MATERIALS AND METHODS Mouse embryonic fibroblast feeder cells preparation CF-1 mice under specific pathogen-free conditions were provided by the Animal Center of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. A mouse at 13.5 days of pregnancy was sacrificed by cervical dislocation, and a fetal mouse obtained under sterile conditions. The head, four limbs and internal organs were removed. The trunk was minced with eye scissors into smaller pieces and mixed with 0.25 and 0.05% trypsin (mixed in equal volume ratio, 10 ml) followed by cultivation for 15 min at 37℃ in a 5% CO2 humidified atmosphere. The culture dish was taken out every 3 to 4 min and the tissues were gently pipetted out for 10 min. An equal volume of mouse embryonic fibroblast (MEF) was added to terminate the reaction. The cells were harvested by centrifugation (200 g, 5 min) and incubated onto 10 cm culture dishes containing 25 ml MEF media followed by cultivation at 37°C in a 5% CO2 humidified atmosphere. Subconfluent MEF cultures were treated with 10 mg/ml Mitomycin C (Sigma, St. Louis, MO) for 2.5 h to arrest cell division, trypsinized, and then incubated in MEF medium. MEF’s of passages 2 to 3 were used as feeders. Retroviral vector The Yamanaka plasmid carrying OCT4, SOX2, KLF4 and C-

MYC gene, EGFP plasmid and retrovirus packaging plasmid were provided by Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. They were extracted with a plasmid extraction kit (QIANGEN) and identified by sequencing (primers are listed in Table 1). A Retrovirus was produced in 293T packaging cells by plasmid co-transfection for 48 h and concentrated by ultracentrifugation. The viral titers were determined by transducing 293T cells (1.5 × 105) with virus supernatant (5 μl). Transduced cells were incubated for 48 h and fixed with 4% paraformaldehyde, counter-stained with 4, 6-diamidino-2-phenylindole (DAPI), and viewed on an inverted fluorescence microscope. The titer was calculated based on the following formula: Viral titers (transducing units/ml; IU/ml) = the number of EGFP positive cells / total cell number÷5 × 1.5 × 105 × 103. The retroviral vector was stored at -80°C. Human iPSCs derivation and culture Human skin biopsies (1 × 2) cm were obtained following appropriate ethical approval by the Institutional Review Board of our hospital and after obtaining informed consent from a patient (aged 63 years) with chronic liver failure, the main symptoms of the patient included fatigue, poor appetite, jaundice and a small amount of ascites (less than 200 ml) without clear pathogenesis, and the main biochemical indicators of the patient are summarized in Table 2.

Tissue fragments were cultured using standardized in-house protocols. Fibroblasts grew out of the tissue fragments and when sufficiently numerous, cells were digested with 0.25% trypsin, seeded into 6-well plate (3 × 104 cell/well), and expanded in a standard fibroblast culture medium (2 ml). After incubated for 24 h, fresh fibroblast culture medium was replaced and meanwhile 2 ml of viral vectors containing OCT4, SOX2, C-MYC and KLF4 were added. One control well was selected and added with viral vectors only containing EGFP gene to test virus infection efficiency. The media was changed on day 3 to defined fetal bovine serum. On day 6, the entire well was trypsinized and passaged onto 10 cm gelatin-coated culture dishes (1 × 104

cell/10 cm) which had been pre-seeded the day before with mitomycin C-inactivated MEF feeder cells. After approximately 21 to 30 days, a few colonies which showed hESC morphology were identified. The human iPS colonies were gently picked into new feeder cells and passaged at 1:3 to 1:4 under knockout serum replacement media. Alkaline phosphatase staining After two human iPSCs lines were expanded to P12 passage, alkaline phosphatase staining was conducted using the Alkaline Phosphatase Staining kit (Millipore) following the manufacturer's instruction.

Academia Journal of Biotechnology; Lu et al. 299 RT-PCR for marker genes When human iPSCs were expanded to P6 to P8 passage, total RNAs were extracted from human iPSCs (n = 8) derived from two cell lines using TRIzol Reagent (TaKaRa). For each sample, 1 to 5 μg of total RNA was reverse transcribed using Reverse Transcription System (TIANGEN). The 25 μl PCR reaction mixture contained Taq enzyme 0.25 μl, 10× PCR buffer (Mg2+plus) 2.5 μl, forward primer (10 μM) 1 μl, reverse primer (10μM) 1 μl, cDNA 1 μl, dNTP mix 2 μl and ddH2O 17.25 μl. PCR conditions were initial denaturation at 95°C for 1 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, 68°C for 40 s and finally 68°C for 5 min. Expression values were normalized to the average expression of the housekeeping gene GAPDH. Table 1 shows the sequences of primers for the human iPSCs spontaneous differentiation analysis. Immunofluorescence for marker proteins Human iPSCs were fixed for 20 to 30 min at 4℃ in 4% paraformaldehyde. Cells were blocked with 10% normal goat serum, 1% bovine serum albumin, and 0.3% tritonX-100 in Dulbecco's phosphate-buffered saline (DPBS) at room temperature for 2 h and subsequently incubated overnight at 4°C with the following primary antibodies: Nanog (Abnova), SSAE-4 (Invitrogen), SSAE-3 (Invitrogen), TRA-1-60 (Invitrogen) and TRA-1-81 (Invitrogen). Cells were then washed thrice in DPBS and incubated with FITC-conjugated secondary antibodies for 1 h at 37°C. Then, 1 μg/ml DAPI was used to stain the cell nuclei. Also, corresponding isotype antibody or the normal serum from the same species with the primary antibody was used as negative control. Karyotype analysis Human iPSC lines (n = 2) during the logarithmic phase of growth were harvested and chromosomal studies performed at Central Hospital, Zhujiang Hospital, Southern Medical University using standard protocols for high resolution G-banding. Teratomas

Human iPSCs (n = 2) were treated with collagenase IV for 10 min at room temperature and collected by centrifugation. Approximately 5 × 106 cells were subcutaneously injected into immune-deficient SCID mice (male, 5-week-old, obtained from the Animal Experiment Center of Southern Medical University). Mice were housed and maintained at 20 to 24°C, 50% room humidity in a 14 h light, 10 h dark cycle with food and water ad libitum. Four to 8 weeks after injection, the mice were sacrificed.

Teratomas were dissected, fixed overnight in 4% paraformaldehyde, and dehydrated through a graded series of alcohols to xylene. The tissue was embedded in paraffin and serially sectioned at 5 μm followed by hematoxylin/eosin (HE) staining and characterization.

Differentiation of iPSCs

Human iPSCs in the logarithmic phase of growth were digested with collagenase IV for 1 h and precipitated for some time. The precipitates were seeded onto 6-well plate and gently re-suspended in hES medium without bFGF. Seven days later, the formed embryonic bodies were transferred to a 0.1% gelatin-treated culture dish and cultured for 2 weeks. Low methylation status was closely related to the cells’ self renewable potential; therefore, to further confirm the differentiation potential of iPSCs, DNA methylation status of which were detected using the bisulfite method.

RESULTS

Generation of human iPSCs in MEF feeder cells

Liver failure patient-derived somatic cells were transduced with four defined factors (OCT4, SOX2, KLF4, and C-MYC). On days 7 to 10, the dermal fibroblast began to make changes in morphology from spinal to round shape. After 2 to 3 weeks of culture in hES cell-supporting conditions, a few compact refractile ES-like colonies emerged among a background of fibroblasts, showing large nucleoli and scant cytoplasm (Park et al., 2007). Some colonies with typical ES-like morphology were selected and further expanded to give rise to stable cell lines (Figure 1).

Characterization of established human iPSCs

Human iPSC lines (n = 2) were stained for alkaline phosphatase expression and the results showed both of them exhibited strong alkaline phosphatase activity (Figure 2). To check the stemness of the iPSC lines (n = 2), the expression of core transcription factors and surface markers including OCT4, SOX2, FGF4, KLF4, ERAS, DAX1, ESG1, NANOG, SSEA4, TRA1-60 and TRA1-81 were tested by RT-PCR or immunofluorescence assay. The results showed that all iPSCs examined expressed hESC markers (Figure 3) and were positive for SSEA-4, TRA-1-60, TRA-1-81, and NANOG (Figure 4). Additionally, all putative human iPSC lines also maintained a normal 46 XY karyotype (Figure 5).

Human iPSCs spontaneously differentiate into cell types of different germ layers

The ability of iPSCs to differentiate into all cell types

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Figure 1. Morphological observation of iPS (×100) a: The first day after transfection; b: The seventh day after transfection; c: The tenth day after transfection; d: The twenty-first day after transfection.

provides the basis for their potential in regenerative medicine. Thus, we examined the pluripotency of iPSCs by teratoma formation. Human iPSC lines (n = 2) were subcutaneously injected into immunodeficient SCID mice. After 2 months, the mice were sacrificed and teratomas dissected to perform HE staining. Histological examination of the teratomas revealed that they differentiated into all three germ layers, including endoderm, mesoderm and ectoderm (Figure 6). We further tested the pluripotency of iPSCs in vitro. Like hESCs, these iPSCs formed embryoid bodies in a suspension culture after two days (Figure 6). After 7 days of growth in suspension for in hESC medium in the absence of bFGF, the embryoid bodies were replanted under adherent conditions for 14 days. The result indicated that the patient-specific human iPSCs are pluripotent, and the embryoid bodies were allowed to spontaneously differentiate (Figure 6). Low methylation status of both NANOG and OTC4 was observed in both C1 and C2 iPCS clone which confirmed that both the iPSC clones had good differentiation potential. Based on these results, we conclude that the iPSCs meet the criteria of hESCs and

could serve as a clinically important source of stem cells. DISCUSSIONS The iPSC technology provides an opportunity to generate cells with characteristics of ES cells, including pluripotency and potentially unlimited self-renewal (Zhao et al., 2008), and thereby, it is expected to be a new potential source of cells for the treatment of many different degenerative diseases, including liver failure. It has been clearly demonstrated that iPSC-Heps can be generated from iPSCs using the stepwise differentiation protocol, and intravenous transplantation of iPSCs can mobilize to the damaged liver area and extensively reduced the hepatic necrotic area, improve liver functions and motor activity, and rescue TAA-treated mice from lethal in mice with TAA-induced liver failure (Chiang et al., 2011). Therefore, research on iPSCs generated from patient with chronic liver failure may provide an inexhaustible cell source for hepatocyte transplantation to patients with end-stage liver disease.

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Figure 2. Alkaline phosphatase staining (×100) a: iPSC-C1; b: iPSC-C2.

In this study, human iPSCs from an adult patient with liver failure by retroviral transduction of OCT4, SOX2, C-MYC and KLF4 with the use of MEF feeder cells culture conditions was successfully generated. We analyzed colonies selected for hES-like morphology, expression of

alkaline phosphatase, SSEA4, TRA-1-60, TRA-1-81 and NANOG, which were all markers shared with hESCs (Adewumi et al., 2007). The established human iPSCs showed the expression of three embryonic germ layers after spontaneous differentiation. Thereby, human iPSCs

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Figure 3. RT-PCR analyses of ES marker genes in iPS cells, ES cells, and HFs. All iPSCs examined (C1 and C2) expressed hESC markers including OCT4, NANOG, SOX, ZFP, ESGL, ERAS, FGF, and DAX. NANOG (both total and endo) and SOX2 endo were enhanced in iPSC and hES cells compared with HF cells, and other marker genes were all expressed in iPSCs and hES cells while not expressed in HFs cells.

may also serve as an inexhaustible cell source for hepatocyte transplantation to patients with end-stage liver disease.

However, this technology is still in its infancy. To realize the full application of iPSCs, it will be essential to improve the methodologies for iPSCs generation and to precisely evaluate each clone and sub-clone of iPSCs for their safety

and efficacy (Yamanaka, 2009). The main strategies for obtaining iPSCs was based on selection of fibroblasts (Takahashi et al., 2007; Liu et al., 2008), keratinocytes (Aasen and Belmonte, 2010) and blood progenitor cells (Raya et al., 2009) as all of them have genetic stability and are easily accessible for reprogramming by simple methods (Sakurai and Yamanaka, 2011). Recently, Ghosh et al.

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Figure 4. Immunofluorescence analyses of specific antigen of hES cells. Scale bar: 100μm. Human iPSCs examined were positive for SSEA-4, TRA-1-60, TRA-1-81, and Nanog.

(2010) compared the transcriptional profiling of human iPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to hESCs. Their

results revealed that fetal fibroblast-derived human iPSCs more closely resemble hESCs, followed by adipose, neonatal fibroblast and keratinocyte-derived human iPSCs

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Figure 5. Karyotype analysis. Human iPS cells (n=2) expressed normal 46 XY karyotypes. a: iPSC-C1; b: iPSC-C2.

(Ghosh et al., 2010). Thus, in this study, we also selected dermal fibroblast from a liver failure patient as target somatic cell to induce the generation of hiPSCs.

Regardless of origin, the reprogramming efficiency of somatic cells is low. For example, human iPSCs have been demonstrated to arise with an efficiency of approximately

0.01% after transduction with four factors in fibroblast and approximately 0.001% after transduction with three factors (Tsai et al., 2010; Wernig et al., 2008).

The efficiency of iPS generation from dermal papilla cells is 1.38% with four factors and greatly reduced to 0.024% with two factors (Tsai et al., 2010). Subsequently, several

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Figure 6. Pluripotency of disease-specific human iPSCs. a: Teratoma formation assay. The representative series of hematoxylin-eosin stained sections from a formalin-fixed teratoma produced from human iPS cell lines. They formed mature, cystic teratomas with tissues representing all three embryonic germ layers including endoderm, mesoderm, and ectoderm (×100); b: Embryoid body formation assay. Embryoid bodies could be formed in suspension culture after 7 days and were allowed to spontaneously differentiate under adherent conditions after 14 days (×100); c: Methylation status of the NANOG gene in disease-specific human iPSCs. Lower methylation status was observed in both C1 and C2 iPCS clone; d: Methylation status of the OCT4 gene in disease-specific human iPSCs. Lower methylation status was observed in both C1 and C2 iPCS clone.

studies suggest the introduction of six transcription factors (Liao et al., 2008), Vitamin C (Esteban et al., 2010), Butyrate (Mali et al., 2010), and some miRNAs (Subramanyam et al., 2011) and all have significantly improved the transduction efficiency. In this study, iPSCs were established by retroviral transduction of four reprogramming factors. Viral vectors containing only the EGFP gene were used to test virus infection efficiency. As a result, EGFP positive cells account for 98 to 100% of human fibroblasts. However, there is insufficient evidence to conclude that all four factors have an effect on EGFP positive cells.

At present, a human ES cell line has not been established, and therefore, there are no specific ES markers for humans.

Although, the teratoma formation test can demonstrate human iPSCs differentiation into three germ layers and possess the potential of multipotential differentiation, it is still not a strict criterion for pluripotent cell identification, and further chimeras formation technology should be developed (Robinton and Daley, 2012). Summary Generation and characterization of iPSCs generated from a dermal fibroblast of an adult patient with chronic liver failure in this study was demonstrated, as the iPSCs are

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Cite this article as: Lu H, Jiang J, Huang Z, Wu Q, Gao Y (2016). Induced Pluripotent Stem Cells Generated from Patients with Chronic Liver Failure. Acad. J. Biotechnol. 4(8): 297-306. Submit your manuscript at http://www.academiapublishing.org/ajb