技术资料

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

BACKGROUND Neuritic degeneration is an important early pathological step in neurodegeneration. AIM The purpose of this study was to explore the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of endoplasmic reticulum (ER) and mitochondria. METHODS Here,we set up neuritic degeneration models by neurite cutting-induced neural degeneration in human-induced pluripotent stem cell-derived neurons. RESULTS We found that neuritic ER becomes fragmented and forms complexes with mitochondria,which induces IP3R-dependent mitochondrial Ca(2+) elevation and dysfunction during neuritic degeneration. Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca(2+) elevation during neuritic degeneration. Mechanically,tightening of the ER-mitochondria associations by expression of a short synthetic linker" and ER Ca(2+) releasing together could promote mitochondrial Ca(2+) elevation� View Publication

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. View Publication

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs),a group of chromatin remodeling enzymes,has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that,as part of mTOR complex 1 (mTORC1),drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2),an mTOR inhibitor,was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes,loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity,and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease. View Publication

Human U1 small nuclear (sn)RNA,required for splicing of pre-mRNA,is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes,also located on chromosome 1 (1q12-21),were thought to be pseudogenes. However,many of these variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells� View Publication

In metazoans,the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes,by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific,while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps,we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover,cLADs are depleted of synteny breakpoints,pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically,the overall sequence conservation is low for cLADs. Instead,cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this A/T rule" in 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

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

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

Regenerative medicine is in need of solid,large animal models as a link between rodents and humans to evaluate the functionality,immunogenicity,and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model,genetically close to humans and readily used worldwide in clinical research. Until now,only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore,we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV,SB431542,PD0325901,and ascorbic acid via a novel,excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4,KLF4,SOX2,C-MYC). Endogenous pluripotency markers like OCT3/4,KLF4,SOX2,C-MYC,LIN28,NANOG,and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors,megakaryocytes,adipocytes,chondrocytes,and osteogenic cells. Thus,all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations. 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