技术资料

The comparative evaluation of different 3D matrices-Matrigel,Puramatrix,and inverted colloidal crystal (ICC) scaffolds-provides a perspective for studying the pathology and potential cures for many blood and bone marrow diseases,and further proves the significance of 3D cultures with direct cell-cell contacts for in vitro mimicry of the human stem cell niche. View Publication

Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere,the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development,we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+] i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease. textcopyright 2013 Elsevier Inc. View Publication

Efficient derivation of neural cells from human embryonic stem cells (hESCs) remains an unmet need for the treatment of neurological disorders. The limiting factors for current methods include being labor-intensive,time-consuming and expensive. In this study,we hypothesize that the substrate topography,with optimal geometry and dimension,can modulate the neural fate of hESCs and enhance the efficiency of differentiation. A multi-architectural chip (MARC) containing fields of topographies varying in geometry and dimension was developed to facilitate high-throughput analysis of topography-induced neural differentiation in vitro. The hESCs were subjected to direct differentiation"� View Publication

Regenerative medicine,relying on human embryonic stem cell (hESC) technology,opens promising new avenues for therapy of many severe diseases. However,this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress,growth rates,and heterogeneous cellular states under current culture conditions. In this study,we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states,precisely control growth rates,significantly increase cell production,and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple,robust,scalable,and suitable for high-throughput screening and drug discovery. View Publication

LIN28 is a conserved RNA-binding protein implicated in pluripotency,reprogramming,and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis,but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28,we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs,reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets,we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. ?? 2012 Elsevier Inc. View Publication

In mice and humans,it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors,Oct3/4,Klf4,Sox2,and c-Myc (OKSM). Here,we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2),leukemia inhibitory factor (LIF),or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4),SOX2,NANOG,and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL,DPPA5,FGF5,REX1,and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo,the ciPSCs created solid tumors,which failed to reach epithelial structure formation,but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance,there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary,we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species,but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown. View Publication

The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell lines from a control subject,a patient with sporadic FTD,and an FTD patient with a novel heterozygous GRN mutation (progranulin [PGRN] S116X). In neurons and microglia differentiated from PGRN S116X induced pluripotent stem cells,the levels of intracellular and secreted PGRN were reduced,establishing patient-specific cellular models of PGRN haploinsufficiency. Through a systematic screen of inducers of cellular stress,we found that PGRN S116X neurons,but not sporadic FTD neurons,exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover,the serine/threonine kinase S6K2,a component of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways,was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by PGRN expression. Our findings identify cell-autonomous,reversible defects in patient neurons with PGRN deficiency,and provide a compelling model for studying PGRN-dependent pathogenic mechanisms and testing potential therapies View Publication

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019),which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization,clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together,our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure. View Publication

Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here,we demonstrate that a recombinant lectin probe,rBC2LCN,a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells,is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in culture medium containing fluorescent dye-labeled rBC2LCN. Fluorescent dye-labeled rBC2LCN was also successfully used to separate live pluripotent stem cells from a mixed cell population by flow cytometry. textcopyright 2013 Elsevier Inc. View Publication

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them,Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT),which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However,they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition,the relationship between the phenotype and mHTT levels has not been elucidated. Herein,we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments,which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ∼10% of mHTT is sufficient to prevent toxicity,whereas ∼90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore,the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.—Lu,B.,Palacino,J. A novel human embryonic stem cell-derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT-dependent neurodegeneration. View Publication

Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context,we explored the growth and differentiation potential of mesenchymal progenitors (MPs) derived in vitro from human embryonic stem cells (hESCs). Similar to MPs isolated from bone marrow,hESC derived MPs (hESC-MPs) efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1,hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5,MEF2C,HAND2 and MYOCD. Nevertheless,NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes,raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that,although hESC-MP derived cells expressed a suite of cardiac related genes,they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes,but treated cells retain a mesenchymal like phenotype. View Publication

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication