技术资料

Mutations in human induced pluripotent stem cells (iPSCs) pose a risk for their clinical use due to preferential reprogramming of mutated founder cell and selection of mutations during maintenance of iPSCs in cell culture. It is unknown,however,if mutations in iPSCs are due to stress associated with oncogene expression during reprogramming. We performed whole exome sequencing of human foreskin fibroblasts and their derived iPSCs at two different passages. We found that in vitro passaging contributed 7% to the iPSC coding point mutation load,and ultradeep amplicon sequencing revealed that 19% of the mutations preexist as rare mutations in the parental fibroblasts suggesting that the remaining 74% of the mutations were acquired during cellular reprogramming. Simulation suggests that the mutation intensity during reprogramming is ninefold higher than the background mutation rate in culture. Thus the factor induced reprogramming stress contributes to a significant proportion of the mutation load of iPSCs. View Publication

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. 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

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

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

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a leading monogenic neurodegenerative disorder affecting premutation carriers of the fragile X (FMR1) gene. To investigate the underlying cellular neuropathology,we produced induced pluripotent stem cell-derived neurons from isogenic subclones of primary fibroblasts of a female premutation carrier,with each subclone bearing exclusively either the normal or the expanded (premutation) form of the FMR1 gene as the active allele. We show that neurons harboring the stably-active,expanded allele (EX-Xa) have reduced postsynaptic density protein 95 protein expression,reduced synaptic puncta density and reduced neurite length. Importantly,such neurons are also functionally abnormal,with calcium transients of higher amplitude and increased frequency than for neurons harboring the normal-active allele. Moreover,a sustained calcium elevation was found in the EX-Xa neurons after glutamate application. By excluding the individual genetic background variation,we have demonstrated neuronal phenotypes directly linked to the FMR1 premutation. Our approach represents a unique isogenic,X-chromosomal epigenetic model to aid the development of targeted therapeutics for FXTAS,and more broadly as a model for the study of common neurodevelopmental (e.g. autism) and neurodegenerative (e.g. Parkinsonism,dementias) disorders. View Publication

Functional cardiomyocytes can be efficiently derived from human pluripotent stem cells (hPSCs),which collectively include embryonic and induced pluripotent stem cells. This cellular platform presents exciting new opportunities for development of pharmacologically relevant in vitro screens to detect cardiotoxicity,validate novel drug candidates in preclinical trials and understand complex congenital cardiovascular disorders,to advance current clinical therapies. Here,we discuss the progress and impediments the field has faced in using hPSC-derived cardiomyocytes for these in vitro applications,and highlight that rigorous protocol optimisation and standardisation,scalability and automation are remaining obstacles for the generation of pure,mature and clinically relevant hPSC cardiomyocytes. View Publication

Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence. 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

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