技术资料

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs,precluding the rational design and optimisation of such platforms. In this study,we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase,combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment,we developed protocols to accurately measure uptake and production rates of metabolites,cell density,growth rate and biomass composition,and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria,however,whilst the results of this study confirmed that glycolysis is indeed highly active,we show that at least in MEL-2 hESC,it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources,such as glutamine to maximise ATP production. Under both conditions,glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect,with high aerobic activity despite high lactate production,challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of View Publication

BACKGROUND:Emerging studies of human pluripotent stem cells (hPSCs) raise new prospects for neurodegenerative disease modeling and cell replacement therapies. Therefore,understanding the mechanisms underlying the commitment of neural progenitor cells (NPCs) is important for the application of hPSCs in neurodegenerative disease therapies. It has been reported that epigenetic modifications of histones play important roles in neural differentiation,but the exact mechanisms in regulating hPSC differentiation towards NPCs are not fully elucidated.RESULTS:We demonstrated that suppression of histone deacetylases (HDACs) promoted the differentiation of hPSCs towards NPCs. Application of HDAC inhibitors (HDACi) increased the expression of neuroectodermal markers and enhanced the neuroectodermal specification once neural differentiation was initiated,thereby leading to more NPC generation. Similarly,the transcriptome analysis showed that HDACi increased the expression levels of ectodermal markers and triggered the NPC differentiation related pathways,while decreasing the expression levels of endodermal and mesodermal markers. Furthermore,we documented that HDAC3 but not HDAC1 or HDAC2 was the critical regulator participating in NPC differentiation,and knockdown of HDAC3's cofactor SMRT exhibited a similar effect as HDAC3 on NPC generation.CONCLUSIONS:Our study reveals that HDACs,especially HDAC3,negatively regulate the differentiation of hPSCs towards NPCs at an earlier stage of neural differentiation. Moreover,HDAC3 might function by forming a repressor complex with its cofactor SMRT during this process. Thus,our findings uncover an important epigenetic mechanism of HDAC3 in the differentiation of hPSCs towards NPCs. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication

Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-),containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands,including interleukin (IL)-8 and growth-related oncogene $\$(GRO$\$),which were developed on the basis of our previous studies. First,we confirmed that IL-8 and/or GRO$\$ independent roles to preserve the phenotype of hPSCs. Then,we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly,CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly,we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF. View Publication

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs),and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here,we report a de novo balanced translocation disruption of TRPC6,a cation channel,in a non-syndromic autistic individual. Using multiple models,such as dental pulp cells,induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models,we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development,morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin,a TRPC6-specific agonist,suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome,revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population,and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together,these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.Molecular Psychiatry advance online publication,11 November 2014; doi:10.1038/mp.2014.141. View Publication

BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression. View Publication

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically,various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods,we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods,which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming,thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

PURPOSE: This first-in-human dose-escalation trial evaluated the safety,tolerability,maximal-tolerated dose (MTD),dose-limiting toxicities (DLT),pharmacokinetics,pharmacodynamics,and preliminary clinical activity of pictilisib (GDC-0941),an oral,potent,and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily,initially on days 1 to 21 every 28 days and later,using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET,and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea,rash,and fatigue,whereas the DLT was grade 3 maculopapular rash (450 mg,2 of 3 patients; 330 mg,1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed textgreater90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC textgreater20 htextperiodcenteredμmol/L. Significant increase in plasma insulin and glucose levels,and textgreater25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria,respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile,on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily. View Publication

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin,+ROCKi/-spin,-ROCKi/+spin,and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions,including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation,elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment,and low-cost scalability,which will directly support automated,large-scale production of hEBs and hESC-derived cells needed for clinical,research,or therapeutic applications. View Publication

Gastric diseases,including peptic ulcer disease and gastric cancer,affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis,and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF,WNT,BMP,retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains,proliferative zones containing LGR5-expressing cells,surface and antral mucous cells,and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease,we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor,activation of signalling and induction of epithelial proliferation. Together,these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. View Publication

For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies. View Publication