技术资料

Reduced expression 1 (REX1) is a widely used pluripotency marker,but little is known about its roles in pluripotency. Here,we show that REX1 is functionally important in the reacquisition and maintenance of pluripotency. REX1-depleted human pluripotent stem cells (hPSCs) lose their self-renewal capacity and full differentiation potential,especially their mesoderm lineage potential. Cyclin B1/B2 expression was found to parallel that of REX1. REX1 positively regulates the transcriptional activity of cyclin B1/B2 through binding to their promoters. REX1 induces the phosphorylation of DRP1 at Ser616 by cyclin B/CDK1,which leads to mitochondrial fission and appears to be important for meeting the high-energy demands of highly glycolytic hPSCs. During reprogramming to pluripotency by defined factors (OCT4,SOX2,KLF4,and c-MYC),the reprogramming kinetics and efficiency are markedly improved by adding REX1 or replacing KLF4 with REX1. These improvements are achieved by lowering reprogramming barriers (growth arrest and apoptosis),by enhancing mitochondrial fission,and by conversion to glycolytic metabolism,dependent on the cyclin B1/B2-DRP1 pathway. Our results show that a novel pluripotency regulator,REX1,is essential for pluripotency and reprogramming. View Publication

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ∼ 44% of the seeded cells were rescued,but an optimized heat shock treatment combined with Ri significantly increased cell survival to ∼ 60%. Mechanistically,our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2,hES3) that were used at higher passages (textgreater 86). In contrast,rapid down regulation of Oct4,Tra-1-60,and SSEA4 was observed for ESI049,a clinically compliant line,used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless,our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. textcopyright 2010 Elsevier B.V. All rights reserved. View Publication

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using non-integrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Result showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in two weeks. Compared to other enrichment strategies such as integrative genetic selection,mitochondria labeling or surface marker cell sorting,this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof-of-concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism. View Publication

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles,but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here,we focused on CDKAL1,linked by GWASs to diabetes. Through transcript profiling,we find that expression of the metallothionein (MT) gene family,also linked by GWASs to diabetes,is significantly downregulated in CDKAL1(-/-) cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression. View Publication

Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening,we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons,seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks,without affecting general cell health. To validate our model,activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model,highly suitable to screen for compounds that modulate TAU aggregation. View Publication

Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However,the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations,and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an AtextgreaterG change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE,control iPSC-RPE cells,and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study,we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations,which can enable positive clinical outcomes. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However,the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here,we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further,we investigated the transcriptional changes in mRNA and miRNA levels,using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs. View Publication