技术资料

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However,the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product,both of which can limit the future clinical application of hESC-ECs. Moreover,to fully understand the beneficial effects of stem cell therapy,investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study,we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray,and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover,our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together,we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes,form functional vessels in vivo,and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. View Publication

Mammalian synthetic biology may provide novel therapeutic strategies,help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet,our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design,construction and screening of synthetic gene networks. To address this problem,here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units,27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept,we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements,genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

Human embryonic stem (hES) cells have enormous potential for clinical applications. However,one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis,which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased,F-actin content and distribution is altered,and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However,exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. View Publication

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies,particularly in view of the diverse methods for derivation and maintenance of these cell lines. However,characterization methods are generally not standardized and many currently used assays are subjective,making dependable and direct comparison of cell lines difficult. In order to address this problem,we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines,derived in two different laboratories using different derivation techniques,as pathogen free,genetically stable,and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control,a crucial element of successful hESC research and clinical translation. View Publication

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore,we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs,RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation,while down-regulated miRNAs such as miR-367,miR-18b,and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development,cell cycle progression,cell death,and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis,eye differentiation and development. View Publication

Human pluripotent stem cells (hPSCs) display a very short G1 phase and rapid proliferation kinetics. Regulation of the cell cycle,which is linked to pluripotency and differentiation,is dependent on the stem cell environment,particularly on culture density. This link has been so far empirical and central to disparities in the growth rates and fractions of self-renewing hPSCs residing in different cycle phases. In this study,hPSC cycle progression in conjunction with proliferation and differentiation were comprehensively investigated for different culture densities. Cell proliferation decelerated significantly at densities beyond 50×10(4) cells/cm(2). Correspondingly,the G1 fraction increased from 25% up to 60% at densities greater than 40×10(4) cells/cm(2) while still hPSC pluripotency marker expression was maintained. In parallel,expression of the cycle inhibitor CDKN1A (p21) was increased,while that of p27 and p53 did not change significantly. After 4 days of culture in an unconditioned medium,greater heterogeneity was noted in the differentiation outcomes and was limited by reducing the density variation. A quantitative model was constructed for self-renewing and differentiating hPSC ensembles to gain a better understanding of the link between culture density,cycle progression,and stem cell state. Results for multiple hPSC lines and medium types corroborated experimental findings. Media commonly used for maintenance of self-renewing hPSCs exhibited the slowest kinetics of induction of differentiation (kdiff),while BMP4 supplementation led to 14-fold higher kdiff values. Spontaneous differentiation in a growth factor-free medium exhibited the largest variation in outcomes at different densities. In conjunction with the quantitative framework,our findings will facilitate rationalizing the selection of cultivation conditions for the generation of stem cell therapeutics. View Publication

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids,rather than two-dimensional (2D) monolayers,is advantageous for many regenerative,biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly,we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells,where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. View Publication

Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2??-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined,these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here,we show that low O2 tension and hypoxiainducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies,we implicate O2 tension as a modifier of a key cell fate decision,namely whether neural progenitors differentiate toward neurons or glia. Furthermore,our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC,a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage.We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types. View Publication

The use of human pluripotent stem cells,including embryonic and induced pluripotent stem cells,in therapeutic applications will require the development of robust,scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs),but challenges specific to hESCs will also have to be addressed,including development of defined,humanized culture media and substrates,monitoring spontaneous differentiation and heterogeneity in the cultures,and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. View Publication

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here,we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology,gene expression,and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and,therefore,offers a promising approach to studying vocal fold biology and biomechanics in health and disease. View Publication