技术资料

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

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

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

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

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here,we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine,which normally induces cardiac hypertrophy,had no additional effects under serum conditions. Likewise,hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype,did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum. View Publication

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells,retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE),and fetal RPE. The protocols include collection of RNA for analysis by microarray,and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally,cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. View Publication

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus,PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression,splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However,when cell nuclei were microirradiated by UV-A,the mobility of PRMT1 cytoplasmic bodies increased,size was reduced,and disappeared within approximately 20 min. The same response occurred after $$-irradiation of the whole cell population,but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together,we show that PRMT1 concentrates in cytoplasmic bodies,which respond to DNA injury in the cell nucleus,and to treatment with various PRMT1 inhibitors. View Publication

Clinical trials are currently used to test therapeutic efficacies for lung cancer,infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA,protein,morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates,volumes and cryoprotectant formulation were optimized to maintain tissue architecture,decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis,showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology,RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types,including alveolar epithelial cells,fibroblasts and stem cells,from the tissue for at least three months after cryopreservation. This new method should provide a uniform,cost-effective approach to the banking of biospecimens,with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. View Publication

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet,cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation,viability and differentiation potential. Here,a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation,mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover,the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics. View Publication

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1,also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution,with,at any given time,a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage,likely under pressure of host restriction factors,which act notably by silencing L1 expression during early embryogenesis. Here,we demonstrate that in human embryonic stem (hES) cells,KAP1 (KRAB [Kruppel-associated box domain]-associated protein 1),the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses,represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice,we documented a similar chronologically conditioned pattern,albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP,suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements,but their younger,human-specific counterparts (L1Hs) were unaffected. Instead,they were stimulated by depleting DNA methyltransferases,consistent with recent evidence demonstrating that the PIWI-piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether,these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected. View Publication