技术资料

Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However,applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here,we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines,we found that mutations in CDKAL1,KCNQ1,and KCNJ11 led to impaired glucose secretion in vitro and in vivo,coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform,which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects,paves the way for precision therapy of metabolic diseases. View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

BACKGROUND: Lineage specific differentiation of human embryonic stem cells (hESCs) is largely mediated by specific growth factors and extracellular matrix molecules. Growth factors initiate a cascade of signals which control gene transcription and cell fate specification. There is a lot of interest in inducing hESCs to an endoderm fate which serves as a pathway towards more functional cell types like the pancreatic cells. Research over the past decade has established several robust pathways for deriving endoderm from hESCs,with the capability of further maturation. However,in our experience,the functional maturity of these endoderm derivatives,specifically to pancreatic lineage,largely depends on specific pathway of endoderm induction. Hence it will be of interest to understand the underlying mechanism mediating such induction and how it is translated to further maturation. In this work we analyze the regulatory interactions mediating different pathways of endoderm induction by identifying co-regulated transcription factors.backslashnbackslashnRESULTS: hESCs were induced towards endoderm using activin A and 4 different growth factors (FGF2 (F),BMP4 (B),PI3KI (P),and WNT3A (W)) and their combinations thereof,resulting in 15 total experimental conditions. At the end of differentiation each condition was analyzed by qRT-PCR for 12 relevant endoderm related transcription factors (TFs). As a first approach,we used hierarchical clustering to identify which growth factor combinations favor up-regulation of different genes. In the next step we identified sets of co-regulated transcription factors using a biclustering algorithm. The high variability of experimental data was addressed by integrating the biclustering formulation with bootstrap re-sampling to identify robust networks of co-regulated transcription factors. Our results show that the transition from early to late endoderm is favored by FGF2 as well as WNT3A treatments under high activin. However,induction of late endoderm markers is relatively favored by WNT3A under high activin.backslashnbackslashnCONCLUSIONS: Use of FGF2,WNT3A or PI3K inhibition with high activin A may serve well in definitive endoderm induction followed by WNT3A specific signaling to direct the definitive endoderm into late endodermal lineages. Other combinations,though still feasible for endoderm induction,appear less promising for pancreatic endoderm specification in our experiments. View Publication

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging,intraoperative visualization,and therapeutic applications. These APC analogs derived from chemically-synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB),APC permeability across intact BBB remains unknown. Three of our APC analogs,CLR1404 (PET radiotracer),CLR1501 (green fluorescence),and CLR1502 (near infrared fluorescence),were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEERtextgreater1500 Ω-cm(2)) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the iPSC-BMEC (35±5.7 (CLR1404),54±3.2 (CLR1501),and 26±4.9 (CLR1502) x10(-5) cm/min) compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29). Only our fluorescent APC analogs (CLR1501,CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover,addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP,and may affect fluorescence-guided applications. Overall,the characterization of APC analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents. View Publication

We had earlier reported the derivation and characterization of two new sibling human embryonic stem cell lines BJNhem19 and BJNhem20,from discarded grade III embryos of Indian origin. We report here the characteristics of the two sibling cell lines after long-term continuous culture for over 2 yr during which they have been passaged over 200 times. We show that both cell lines adapt well to culture on various mouse and human feeders as well as in feeder-free conditions. The cells show normal diploid karyotype and continue to express all pluripotency markers. Both cell lines differentiate to derivatives of all three germ layers in vitro. However as reported earlier,BJNhem19 is unable to generate teratomas in nude or SCID mice or differentiate to beating cardiomyocytes when tested over several passages during long-term stable culture. On the other hand,the cardiac differentiation capacity of BJNhem20 is greatly increased,and it can generate beating cardiomyocytes that proliferate when isolated and cultured further. In conclusion,the two cell lines have maintained a stable phenotype for over 2 yr and are indeed immortal. Their derivation from grade III embryos does not seem to have any adverse effect on their long-term phenotype. The cells can be obtained for research purposes from the UK Stem Cell Bank and from the authors. View Publication

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network,pathway,and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals,highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders,such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome,we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs,both parental alleles are present,distinguishable by the mutation,and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation,in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein. View Publication

Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults,respectively. In both of these cases,a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue,wound healing complications,and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need,we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures,degradation rates,and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro,silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling. View Publication