技术资料

Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors,Oct4,Sox2,Klf4 and c-Myc,also known as the Yamanaka factors. In practice,initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology,but often may go on to fail subsequent assays,such as the alkaline phosphate (AP) assay. In this study,we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly,we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system,and then transferred to a feeder layer for expansion. Furthermore,colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies,45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies,while colonies screened by the feeder-free system were 84% AP+ colonies,16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers,OCT4,SOX2,NANOG,TRA-1-60,TRA-1-81,SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study,we report a simplistic,one-step method for selection of AP+ AF-iPS cells via feeder-free screening. View Publication

Forced expression of selected transcription factors can transform somatic cells into embryonic stem cell (ESC)-like cells,termed induced pluripotent stem cells (iPSCs). There is no consensus regarding the preferred tissue from which to harvest donor cells for reprogramming into iPSCs,and some donor cell types may be more prone than others to accumulation of epigenetic imprints and somatic cell mutations. Here,we present a simple,reproducible,noninvasive method for generating human iPSCs from renal tubular cells present in urine. This procedure eliminates many problems associated with other protocols,and the resulting iPSCs display an excellent ability to differentiate. These data suggest that urine may be a preferred source for generating iPSCs. View Publication

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings,however,requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study,we show that once specified to neuro-epithelial lineage,hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However,our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes,namely,migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes,a new potential source for therapeutic use for brain tumors and other neurological diseases. View Publication

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells,free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death,which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore,we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly,three compounds currently used as clinically approved drugs,nortriptyline,benzethonium chloride and methylbenzethonium chloride,induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs,whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline,a tricyclic antidepressant,has also been described as a potent inhibitor of mitochondrial permeability transition,one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline,although with a different behavior. Our results indicate differential sensitivity of ESCs,hiPSCs and fibroblasts to certain chemical compounds,which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases. View Publication

Somatic cell nuclear transfer,cell fusion,or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors,Brn2 (also known as Pou3f2),Ascl1 and Myt1l,can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1,these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers,even after downregulation of the exogenous transcription factors. Importantly,the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells,as well as pluripotent stem cells,can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine. View Publication

RATIONALE: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis,the key process underlying excitation-contraction coupling. OBJECTIVE: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). METHODS AND RESULTS: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes,spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore,in contrast with hESC-derived cardiomyocytes,ryanodine did not reduce the amplitudes,maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition,spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2),sacroplasmic recticulum calcium-ATPase (SERCA),junction (Jun) and triadin (TRDN),were significantly lower in hiPSC than in hESCs. CONCLUSIONS: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. View Publication

The use of small molecules to 'chemically direct' differentiation represents a powerful approach to promote specification of embryonic stem cells (ESCs) towards particular functional cell types for use in regenerative medicine and pharmaceutical applications. Here,we demonstrate a novel route for chemically directed differentiation of human ESCs (hESCs) into definitive endoderm (DE) exploiting a selective small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3). This GSK-3 inhibitor,termed 1m,when used as the only supplement to a chemically defined feeder-free culture system,effectively promoted differentiation of ESC lines towards primitive streak (PS),mesoderm and DE. This contrasts with the role of GSK-3 in murine ESCs,where GSK-3 inhibition promotes pluripotency. Interestingly,1m-mediated induction of differentiation involved transient NODAL expression and Nodal signalling. Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics,that is expressing α-fetoprotein and HNF4α. Furthermore,1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin. These findings describe,for the first time,the utility of GSK-3 inhibition,in a chemically directed approach,to a method of DE generation that is robust,potentially scalable and applicable to different hESC lines. View Publication

Human pluripotent stem cells (PSCs),encompassing embryonic stem cells and induced pluripotent stem cells,proliferate extensively and differentiate into virtually any desired cell type. PSCs endow regenerative medicine with an unlimited source of replacement cells suitable for human therapy. Several hurdles must be carefully addressed in PSC research before these theoretical possibilities are translated into clinical applications. These obstacles are: (1) cell proliferation; (2) cell differentiation; (3) genetic integrity; (4) allogenicity; and (5) ethical issues. We discuss these issues and underline the fact that the answers to these questions lie in a better understanding of the biology of PSCs. To contribute to this aim,we have developed a free online expression atlas,Amazonia!,that displays for each human gene a virtual northern blot for PSC samples and adult tissues (http://www.amazonia.transcriptome.eu). View Publication

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis,we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably,we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media,up to 80% of cells expressed the neural stem cell marker nestin,and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation,we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together,our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein. View Publication

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication

Disentangling the complex interactions that govern stem cell fate choices of self-renewal,differentiation,or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632,HA-1077,and H-8 all strongly inhibit the kinases ROCK and PRK2,highlighting the important role of these kinases in EC cell survival. Two molecules,GF109203x and rottlerin,induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells,caused the cell cycle arrest,and repressed the expression of pluripotency-associated genes. View Publication