技术资料

Induced pluripotent stem cells (iPSCs) derived from patients with neurodegenerative disease generally lack neuropathological confirmation,the gold standard for disease classification and grading of severity. The use of tissue with a definitive neuropathological diagnosis would be an ideal source for iPSCs. The challenge to this approach is that the majority of biobanked brain tissue was not meant for growing live cells,and thus was not frozen in the presence of cryoprotectants such as DMSO. PMID: 24398250 View Publication

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However,standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium,imposing a significant obstacle to clinical translation. Here,we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions,we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently,transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal,normal karyotype and pluripotency,as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly,we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View Publication

Ring chromosomes are structural aberrations commonly associated with birth defects,mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome,and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes,no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division,ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations,enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of /`chromosome therapy/' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition,our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control,which is of critical relevance to human development and disease. View Publication

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here,iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4,and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry,RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery. ?? 2013. View Publication

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells,chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG,SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture,greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9,type II collagen,and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen,type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state,suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects,and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs. ?? 2014 Elsevier Ltd. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive,result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs,we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate,formulated as a hypertonic solution,gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium,retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together,this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology,high levels (textgreater80%) of pluripotency markers OCT4,SSEA-4,TRA-1-60 andTRA-1-81,a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers,both in vivo and in vitro. View Publication

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy,drug screening,physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods,techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results,we adopted a polycistronic cassette encoding Thomson's cocktail OCT4,NANOG,SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones,based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes,ONSL and OCT4,SOX2,KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly,in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM,we indeed observed a remarkable synergy,yielding a reprogramming efficiency of textgreater2%. We present here a drastic improvement of the reprogramming efficiency,which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore,non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency. View Publication

The ability of human embryonic stem cells and induced pluripotent stem cells to differentiate into all adult cell types greatly facilitates the study of human development,disease pathogenesis,and the generation of screening systems to identify novel therapeutic agents. Autologous cell therapies based on patient-derived induced pluripotent stem cells also hold great promise for the treatment and correction of many inherited and acquired diseases. The full potential of human pluripotent stem cells can be unleashed by genetically modifying a chosen locus with minimal impact on the remaining genome,which can be achieved by targeting genes by homologous recombination. This chapter will describe a protocol for gene modification of pluripotent stem cells by homologous recombination and several methods for the screening and identification of successfully modified clones. View Publication