技术资料

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here,we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells,p53 in hESCs is maintained at low levels in the nucleus,albeit in a deacetylated,inactive state. In response to retinoic acid,CBP/p300 acetylates p53 at lysine 373,which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel,p53 activates expression of miR-34a and miR-145,which in turn repress stem cell factors OCT4,KLF4,LIN28A,and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation,whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs,independently of retinoic acid. Ectopic expression of p53R175H,a mutated form of p53 that does not bind DNA or regulate transcription,failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. View Publication

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However,no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work,we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF,EGF and PDGF. These cells are SSEA4(+),OCT3/4(+),NANOG(+),SOX2(+),LIN28(+),CD13(+),CD105(+),CD34(-),CD45(-),CD90(+),CD29(+),CD73(+),STRO1(+) and CD146(-),and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly,DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm,endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4,GATA6,MIXL1,NANOG,OCT3/4,SOX1 and SOX2 to determine the degree of similarity between DPPSCs,EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs,hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages,they represent an easily accessible source of stem cells,which opens a range of new possibilities for regenerative medicine. View Publication

The study of the regulatory signaling hierarchies of human heart development is limited by a lack of model systems that can reproduce the precise developmental events that occur during human embryogenesis. The advent of human pluripotent stem cell (hPSC) technology and robust cardiac differentiation methods affords a unique opportunity to monitor the full course of cardiac induction in vitro. Here,we show that stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a monolayer-based differentiation protocol that used transforming growth factor β superfamily ligands to generate cardiomyocytes. However,insulin did not repress cardiomyocyte differentiation in a defined protocol that used small molecule regulators of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation,we determined that the inhibitory effects by insulin required Wnt/β-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/β-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus,insulin and Wnt/β-catenin signaling pathways,as a network,coordinate to influence hPSC differentiation to cardiomyocytes,with the Wnt/β-catenin pathway dominant to the insulin pathway. Our study contributes to the understanding of the regulatory hierarchies of human cardiomyocyte differentiation and has implications for modeling human heart development. View Publication

Clinical implications of induced pluripotent stem (iPS) cell technology are enormous for personalized medicine. However,extensive use of viral approach for ectopic expression of reprogramming factors is a major hurdle in realization of its true potential. Non-viral methods for making iPS cells,although plausible,are impractical because of high cost. MicroRNAs are important cellular modulators that have been shown to rival transcription factors and are important players in embryonic development. We have generated distinct microRNA-omes" signature of iPS cells that remain in a near embryonic stem (ES) cell state and distinct from differentiated cells. Recent advances in the microRNA field and experimentally validated microRNAs warrant a review in experimental protocols for microRNA expression profile." View Publication

Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study,we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide),class III (sotalol and amiodarone),and class IV (verapamil),whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability,class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs,verapamil shortened cFPD and FPmin by 25 and 50%,respectively. Furthermore,verapamil reduced beating frequencies drastically. Importantly,the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes,drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine,flecainide,and sotalol was demonstrated at supraphysiological concentrations. Furthermore,off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine. View Publication

Human embryonic stem cells (hESC) are difficult to adapt to 96-well plate assays,such as the MTT assay,because they survive best when plated as colonies,which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first,ROCK inhibitor (ROCKi) was used,which allows accurate counting and plating of single hESC. In the second,small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96-well plate,vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96-well plate assay. View Publication

Human induced pluripotent stem cells (HIPSC) have tremendous value as a source of autologous cells for cellular transplantation in the treatment of degenerative diseases. The protocols described here address methods for large-scale genetic modification of HIPSCs. The first is an optimized method for transfecting HIPSCs cultured in feeder-free conditions. The second method allows nucleofection of trypsinized HIPSCs at an optimal cell density. Both methods enable robust generation of stable HIPSC transfectants within two weeks. Our protocols are highly reproducible and do not require optimization for individual HIPSC and human embryonic stem cell (HESC) lines. View Publication

Pluripotent cells represent a powerful tool for tissue regeneration,but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study,we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated,bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting,we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate,thereby allowing for functional engraftment of pluripotent cells into regenerating tissue. View Publication

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs),we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested,only Sox17,a gene encoding a transcription factor of the SOX family,promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However,they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis,hematopoiesis,and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs,thereby regulating hematopoietic development from hESCs/iPSCs. View Publication

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated,pluripotent cells. The development of defined and feeder-independent culture media has provided a platform for greater reproducibility and standardization in this field. Here we provide detailed protocols for the use of mTeSR™1 and TeSR™2 with various cell culture matrices as well as defined cryopreservation protocols for human embryonic and human induced pluripotent stem cells. View Publication

The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns),nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound,PluriSIn 1,induces ER stress,protein synthesis attenuation,and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1),the key enzyme in oleic acid biosynthesis,revealing a unique role for lipid metabolism in hPSCs. PluriSIn 1 was also cytotoxic to mouse blastocysts,indicating that the dependence on oleate is inherent to the pluripotent state. Finally,application of PluriSIn 1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments. ?? 2013 Elsevier Inc. View Publication

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed,we report that the cells survive and differentiate upon transplant into newborn rat brain. View Publication