技术资料

The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood,limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest,differentiation and cell death. Conversely,inactivation of the entire RB family (RB,p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells,mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally,loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells,uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells. View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

Heterogeneity is an often unappreciated characteristic of stem cell populations yet its importance in fate determination is becoming increasingly evident. Although gene expression noise has received greater attention as a source of non-genetic heterogeneity,the effects of stochastic partitioning of cellular material during mitosis on population variability have not been researched to date. We examined self-renewing human embryonic stem cells (hESCs),which typically exhibit a dispersed distribution of the pluripotency marker NANOG. In conjunction with our experiments,a multiscale cell population balance equation (PBE) model was constructed accounting for transcriptional noise and stochastic partitioning at division as sources of population heterogeneity. Cultured hESCs maintained time-invariant profiles of size and NANOG expression and the data were utilized for parameter estimation. Contributions from both sources considered in this study were significant on the NANOG profile,although elimination of the gene expression noise resulted in greater changes in the dispersion of the NANOG distribution. Moreover,blocking of division by treating hESCs with nocodazole or colcemid led to a 39% increase in the average NANOG content and over 68% of the cells had higher NANOG level than the mean NANOG expression of untreated cells. Model predictions,which were in excellent agreement with these findings,revealed that stochastic partitioning accounted for 17% of the total noise in the NANOG profile of self-renewing hESCs. The computational framework developed in this study will aid in gaining a deeper understanding of how pluripotent stem/progenitor cells orchestrate processes such as gene expression and proliferation for maintaining their pluripotency or differentiating along particular lineages. Such models will be essential in designing and optimizing efficient differentiation strategies and bioprocesses for the production of therapeutically suitable stem cell progeny. View Publication

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background,yet the landscapes of in vivo tissues remain largely uncharted. Here,we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages,lineages,and cellular environments. They also reveal global features of the epigenome,related to nuclear architecture,that also vary across cellular phenotypes. Specifically,developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture,and discuss their implications for lineage fidelity,cellular senescence,and reprogramming. ?? 2013 Elsevier Inc. View Publication

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development,a basement membrane surrounds the neural plate that forms a tight,apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium,in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia. View Publication

Magnesium alloys have attracted great interest for medical applications due to their unique biodegradable capability and desirable mechanical properties. When designed for medical applications,these alloys must have suitable degradation properties,i.e.,their degradation rate should not exceed the rate at which the degradation products can be excreted from the body. Cellular responses and tissue integration around the Mg-based implants are critical for clinical success. Four magnesium–zinc–strontium (ZSr41) alloys were developed in this study. The degradation properties of the ZSr41 alloys and their cytocompatibility were studied using an in vitro human embryonic stem cell (hESC) model due to the greater sensitivity of hESCs to known toxicants which allows to potentially detect toxicological effects of new biomaterials at an early stage. Four distinct ZSr41 alloys with 4 wt% zinc and a series of strontium compositions (0.15,0.5,1,and 1.5 wt% Sr) were produced through metallurgical processing. Their degradation was characterized by measuring total mass loss of samples and pH change in the cell culture media. The concentration of Mg ions released from ZSr41 alloy into the cell culture media was analyzed using inductively coupled plasma atomic emission spectroscopy. Surface microstructure and composition before and after culturing with hESCs were characterized using field emission scanning electron microscopy and energy dispersive X-ray spectroscopy. Pure Mg was used as a control during cell culture studies. Results indicated that the Mg–Zn–Sr alloy with 0.15 wt% Sr provided slower degradation and improved cytocompatibility as compared with pure Mg control. View Publication

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus,hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these,37 hiPSC lines were generated from fetal hepatocytes,2 hiPSC lines from normal hepatocytes,and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome,type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression,flow cytometry,immunocytochemistry,and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors,while fetal hepatocytes could be reprogrammed with three (OCT4,SOX2,NANOG) or four factors (OCT4,SOX2,NANOG,LIN28 or OCT4,SOX2,KLF4,C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes,although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs,the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8),which are expressed in simple epithelial cells,while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose,development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore,bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here,we report identification of small molecules,some of which were neurotransmitter antagonists (trimipramine and ethopropazine),which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers,had a normal karyotype after 20 passages,and could differentiate into all three germ layers. ?? 2013 Elsevier Inc. View Publication

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA),known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts,the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA,resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones,improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus,mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny,and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. View Publication

Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here,we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells,human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h,the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell–cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels,inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity,highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary,we have developed microengineered three-dimensional vascular structures that can be used as a model to test the effects of drugs and study the interaction between different human vascular cell types. In the future,the model may be integrated into larger tissue constructs to advance the development of organs-on-chips. View Publication