技术资料

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy,in vitro modeling of human disease,and drug screening. To date,most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively,transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector,which includes the coding sequences of human OCT4,SOX2,KLF4,and cMYC linked with picornaviral 2A plasmids. Moreover,after reprogramming has been achieved,this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. View Publication

The potential for human disease treatment using human pluripotent stem cells,including embryonic stem cells and induced pluripotent stem cells (iPSCs),also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies,which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study,a comparison of DNA repair pathways in pluripotent cells,as compared to those in non-pluripotent cells,demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair,we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells,while differentiated cells lacked response to this stimulus,and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition,the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype,but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together,these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines,in order to characterize their genomic stability,prior to their pre-clinical and clinical use. View Publication

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan,a 61-amino acid vasodilatory peptide,specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells,little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study,we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1,we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC,the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells,as demonstrated by WST-8 analysis,annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly,immunofluorescence,western blot analysis,real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover,karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary,these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. View Publication

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development,we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,then into replicating Nkx2.1+ lung endoderm,and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,FGF,and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication

Although distinct human induced pluripotent stem cell (hiPSC) lines can display considerable epigenetic variation,it has been unclear whether such variability impacts their utility for disease modeling. Here,we show that although low-passage female hiPSCs retain the inactive X chromosome of the somatic cell they are derived from,over time in culture they undergo an erosion" of X chromosome inactivation (XCI). This erosion of XCI is characterized by loss of XIST expression and foci of H3-K27-trimethylation� View Publication

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However,to date,derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors,which,upon transplantation into dystrophic muscle,are able to engraft efficiently,producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly,transplanted cells also seed the muscle satellite cell compartment,and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies. View Publication

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated,turning MSCs into variety of cell lineages of mesodermal,ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial,these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently,using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function,as well as with specific signalling pathways that promote neural fate commitment,we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However,the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs,comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs,accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright textcopyright 2012 John Wiley & Sons,Ltd. View Publication

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study,we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that,due to the absence of p21/Waf1/Cip1,cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage,the majority of hESCs succumb to apoptosis; however,a subpopulation continues to proliferate,carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that,due to deficient DNA damage response,the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs,paving the way for further major genetic changes. View Publication

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However,due to risks of random integration of the reprogramming transgenes into the host genome,the low efficiency of the process,and the potential risk of virally induced tumorigenicity,alternative methods have been developed to generate pluripotent cells using nonintegrating systems,albeit with limited success. Here,we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors,by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion,they maintain genetic stability,protein level expression of key pluripotency factors,high cell-division kinetics,telomerase activity,repression of X-inactivation,and capacity to differentiate into lineages of the three germ layers,such as definitive endoderm,hepatocytes,bone,fat,cartilage,neurons,and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies,pharmaceutical screening,and disease modeling. View Publication

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however,their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC),human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC),and compared their in-vitro tube formation,migration and cytokine expression profiles,and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless,BM-EC,hESC-EC and hiPSC-EC exhibited typical cobblestone morphology,had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein,and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (Ptextgreater0.05). While increased expression of major angiogenic factors including epidermal growth factor,hepatocyte growth factor,vascular endothelial growth factor,placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (Ptextless0.05),the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (Ptextless0.05). Compared with medium,transplanting BM-EC (n = 6),HUVEC (n = 6),hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion,functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases,and hESC-EC or iPSC-EC are readily available as off-the-shelf" format for the treatment of tissue ischemia." View Publication