技术资料

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

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

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

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

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

Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible,it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus,a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors,derived from human embryonic stem cells,express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells,cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state,during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells. View Publication

Introduction: Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However,hESC are difficult to adapt to 96-well plate screening assays,because they survive best when plated as colonies,which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. Methods: We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy,and the method was tested using known toxicants. Results: The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable,and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products,which contain a variety of chemicals. The screen was done using single wells/dose,and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement,and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied,and methods to monitor and avoid vapor effects were incorporated into the assay. Discussion: Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development. ?? 2012 Elsevier Inc. View Publication

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however,inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study,we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization,the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance,only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel,contained no vitronectin but did contain collagen XII,ig-h3 and novel for hESC-supporting human matrices,substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however,distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus,human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. View Publication

Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency,while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development,respectively. However,how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here,we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers,while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling,hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast,inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage,although cells were still able to activate LEFTY1 and NODAL,direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment,although the expression of NANOG,but not POU5F1,was restored immediately upon Activin A treatment. Thus,downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling. View Publication

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist,Noggin,and the small molecule SB431542,respectively,induces efficient neuralization of hiPSCs,a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost,consistent activity,and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1,a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration,we could selectively modulate the number of SOX1 expressing cells,whereas PAX6,another neural precursor marker,remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations,therefore,suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons,a subset of which also express tyrosine hydroxylase. Thus,the combined use of DMH1,a highly specific BMP-pathway inhibitor,and SB431542,a TGF-β1-pathway specific inhibitor,provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. View Publication

Neural differentiation of human embryonic (ES) and induced pluripotent (iPS) stem cell lines has been used for research in early human development,drug discovery,and cell replacement therapies. It is critical to establish generic differentiation protocols to compare the neural specification potential of each individually derived pluripotent stem cell line and identify the efficacious lines for research and therapeutic use. Here,we describe a reproducible and quantitative protocol to assess the neural progenitor (NP) generation of human pluripotent stem cell lines. This method includes a robust and well-defined neural inducing platform for Pax6(+) neural rosette (neuroectodermal cells) generation,propagation,and subsequent differentiation into nestin(+) NPs. A side-by-side comparison under common culture conditions among three human ES cell lines,TE03,TE06,and BG01V,and one iPS cell line,HD02,showed highly variable efficiency in their differentiation into NPs. View Publication

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent,thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes,which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages,starting with endoderm,which can develop into several organs,including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A in combination with several growth factors. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition,which can be achieved in vitro by addition of cyclopamine. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation. Although successful,this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation,which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation. The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture,herein we present our data with rat heart microvascular endothelial Cells (RHMVEC),primarily for the ease of analysis. View Publication