| Background and ObjectiveHemophilia A (HA), characterized by deficiency of coagulation VIII (FVIII), is an X-chromosome-linked recessive bleeding disorder with an incidence of1in5000males. The clinical manifestation of this disease is unpredictable, recurrent, and spontaneous bleeding in various areas, including soft tissues, major joints, and occasionally in internal organs. Severe hemophilia A (<1%factor activity) is characterized by spontaneous and prolonged hemorrhage that can result in disability and death. A variety of different mutations in the FVIII gene, i.e., point mutations, deletions, insertions, and rearrangements/inversions, have been identified. The intron22inversion of the FVIII gene, causing severe haemophilia A and found in45-50%of patients with severe disease, is the most common gene defect.Current therapies include fixed-dose FVIII prophylaxis, factor replacement therapies, and most recently, gene therapy and cell therapy. Prophylaxis and FVIII replacement therapies are limited by incomplete efficacy, high cost, restricted availability, and the possible development of neutralizing antibodies in chronically treated individuals. Hemophilia A has been recognized as a suitable disease for gene therapy. Expression of only a small percentage of wild-type factor Ⅷ, no need in tissue specificity, can be beneficial to the patient, and it has a broad therapeutic index. The overarching goal of gene therapy is to correct the defective gene sequence, approaching a phenotypic correction of the disease. Addition of exogenous genetic material through viral vectors has been the usual therapeutic approach, which supplements an insufficiency rather than replacing the defective copy of the endogenous gene. Limited success has been reported using gene therapy for the treatment of hemophilia with several phase Ⅰ clinical trials in humans to be finished. The failure is primarily caused by host immune responses to virally encoded proteins and lack of sustained production of the therapeutic gene products. The major goal of cell therapy is transplantation of healthy cells to repair organ damage or replace deficient functions. But the problems of the cell source for transplantation and immune rejection are difficult to be solved.There are some of HA animal models, such as canine model, mice model, sheep model and primate model. A substantial amount of the current knowledge about the mechanisms underlying haemophilia A has been derived from the study of animal models. However, these advantages can be offset by species-specific differences between animals and humans in their biochemical, physiological and anatomical characteristics. Furthermore, human tissues other than blood are usually unavailable in sufficient amounts for research, and animal models of human disease created by disrupting the gene underlying the disorder using gene targeting do not reliably reproduce the human phenotypes. Many drugs that work in animals have not performed well in human clinical trials.Yamanaka and others have reported techniques for generating induced pluripotent stem cells (iPSCs) from adult cells such as fibroblasts. Methods for creating iPS cells that circumvent the need for human embryos should quell the political controversy arising from opposition to stem cell research based on moral and religious premises. IPS cells have the same features as embryonic stem (ES) cells and are capable of self-renewal and differentiation into all adult cell types. Several criteria concerning the generation and potential use of human iPSCs for future research, diagnosis and cell therapy have to be fulfilled. The potential utility of the iPSCs is as model system for research work, cell therapy, drug discovery and for in vitro human pathology modelling. The most attractive application would be the production of patient-specific donor cells for cell replacement and/or tissue substitution. IPS cells can be derived from the patient’s somatic cells, thus avoiding potential side effects related to immune rejection. As a result, such patient-derived iPS cell therapy potentially could function better when implanted in diseased organs than either currently available ES cell lines or gene therapies. Recently, as first time in haemophilia, by generating iPS cells that express wild-type endogenous FVIII protein and transplanting them into the hepatic parenchyma, Xu et al. demonstrated an effective phenotypic correction of hemophilia, providing additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future. Therefore, iPSCs technology could represent a potential alternative based on cellular therapy, because there are not relevant results for phenotypic correction in human haemophilia. Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a new unique opportunity for studying HA.Here we described the successful generation of iPSCs from a Chinese patient with hemophilia A that bears the intron22inversion mutation in the coagulation factor Ⅷ gene. These iPSCs were produced by oriP/EBNAl episomal vectors over-expressing OCT4, SOX2, KLF4and SV40LT. Furthermore, we differentiated the HA iPSCs into hepatocyte-like cells and investigated and characterized the disease phenotype in HA iPSCs-derived-hepatocytes.Methods1. The establishment of integration-free HA iPSCsUrine cells were obtained from one severe HA patient. Under feeder-free and xeno-free conditions, HA urine cells were reprogrammed to iPSCs using oriP/EBNA1episomal vectors that express OCT4, SOX2, KLF4and SV40LT. The use of microRNA clusters302-367enhanced somatic cell reprogramming. AP staining, transgene integration, karyotyping, STR analysis, bisulfate sequencing, immunofluorescence, flow cytometry, EB differentiation and teratoma assay were performed to identify and characterize HA iPSCs.2. Hepatic differentiation of HA iPSCsUnder serum-free and monolayer conditions, we differentiated HA iPSCs into hepatocyte-like cell through3stages, i.e., definitive endoderm, hepatic progenitors, and hepatocyte-like cells, generally simulating normal liver development in vivo. Appearance of markers corresponding to the3phases was verified by the observation with immunofluorescence microscopy. In addition, the accumulation of glycogen by periodic acid Schiff (PAS) staining was also observed.3. Characterization of the disease phenotype in HA iPSCs-derived-hepatocytesThe expression of FⅧ gene of HA iPSCs-derived-hepatocytes was detected by qPCR analysis and immunofluorescence microscopy. FⅧ activity assay was used to identify the coagulation function of HA iPSCs-derived-hepatocytes.Results1. The establishment of integration-free HA iPSCs15iPS cell lines were generated and2iPSC clones were selected for further characterization. These HA iPSCs were positive for alkaline phosphatase activity and expressed pluripotent markers including SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, OCT4and NANOG, as assessed by immunofluorescence microscopy and flow cytometry. The absence of the vector and transgene sequences in the genomic DNA of HA iPSCs was verified by PCR and RT-PCR, showing that exogenous transgenes expression was silenced. LD-PCR confirmed the intron22inversion mutation in the coagulation factor Ⅷ gene. The karyotype was normal and OCT4and NANOG proximal promoters were demethylated. Moreover, short tandem repeat (STR) analysis confirmed that HA iPSCs are originated from HA donor urine cells and not contamination with other cell lines grown in our laboratory. In addition, their pluripotent properties were assessed by embryoid body (EB) and teratoma formation, both of which produced derivatives of the3germ layers including rather complex structures in the case of teratomas. Therefore, we have successfully reprogrammed HA urine cells into cells that are pluripotent and stably display human ESCs-like characteristics. 2. Hepatic differentiation of HA iPSCsWe differentiated HA iPSCs into hepatocyte-like cells. At different stages of differentiation, the differentiated cells of HA iPSCs expressed the corresponding markers:the definitive endoderm markers (SOX17and FOXA2), the hepatic progenitors markers (HNF4a and AFP), and the hepatocyte-like cells markers (A1AT and ALB). Further function assays showed that HA iPSCs-derived-hepatocytes had glycogenesis capability.3. Disease modeling with HA iPSCsCompared with normal iPSCs-derived-hepatocytes or ESCs-derived-hepatocytes, HA iPSCs-derived-hepatocytes showed disease phenotype of failed FVIII gene transcript running and deficient FVIII protein expression. FVIII activity of HA iPSCs-derived-hepatocytes was significantly lower than that of normal controls. In conclusion, we established cellular disease model of HA with iPSCs.Conclusions1. HA patient’s urine cells can be reprogrammed to induced pluripotent stem cells without viral vectors and transgene sequences integration.2. Hepatocyte-like cells can be efficiently derived from HA iPSCs in defined, serum free conditions in monolayer induction by simulating the liver development in vivo.3. HA iPSCs-derived-hepatocytes showed disease phenotype of defective coagulation function of FⅧ. Disease modeling with HA iPSCs sets up a new platform to investigate the underlying mechanisms of HA and explore optimal strategy for personalized medicine. |
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