技术资料

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

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

UNLABELLED Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model,then epigenetic modulation might enrich the CSC population,thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis,primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB),a potent DNA methyltransferase-1 inhibitor,and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal,superior tumor-initiating capacity in serial transplantations,and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes,although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g.,EGFR,SRC,and MYC). The common CSC signature was associated with malignant progression,which is enriched in poorly differentiated tumors,and was highly predictive of prognosis in liver and other cancers. CONCLUSION Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs. View Publication

In human B-acute lymphoblastic leukemia (B-ALL),RAG1-induced genomic alterations are important for disease progression. However,given that biallelic loss of the RAG1 locus is observed in a subset of cases,RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model,we identified a new,Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM,a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. View Publication

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin�?� CD45�?� (VE-cad�?�CD45�?�) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study,we developed a new coaggregation culture system,which supports maturation of a novel population of CD45-negative (VE-cad�?�CD45�?�CD41�?�) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad�?�CD45�?� pre-HSCs (termed here type I and type II pre-HSCs,respectively),thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta,we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers,in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs,in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal. View Publication

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study,we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2,lipoprotein lipase,and adipsin increased during adipocytogenesis of hMSCs. Simultaneously,the expression of canonical WNT2,10B,13,and 14 decreased,whereas non-canonical WNT4 and 11 increased,and WNT5A was unchanged. A small molecule WNT mimetic,SB-216763,increased accumulation of β-catenin protein,inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast,knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells. View Publication

Pluripotent stem cells,both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC),can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However,the tumorigenic potential of these cells remains a great concern,as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice,most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal,cardiac,or endothelial cells prior to human transplantation,drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study,we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells,and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer,whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall,our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation,and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy. View Publication

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep,together with fluorescently tagged streptavidin,was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First,approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin,whereas only background fluorescence was detected using spleen cells from untreated mice. In addition,Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells,and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third,Ep-receptor expression was found to correlate positively with expression of transferrin receptors,confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations,for elucidating the structure of the Ep-receptor,and for studying the biological activities of Ep at the cellular and molecular level. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay,we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives,but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response,revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK,a key NHEJ component,by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast,NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus,DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells. View Publication

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population,but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation,increased ability of cells to form mammospheres,and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells,which express high basal levels of TG2,shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together,these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells. View Publication

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