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

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

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

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

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived,patient-specific pluripotent cells for use in cell-based regenerative therapies. However,current methods of cell culture are tedious and expensive,and the mechanisms underlying cell proliferation are not understood. In this study,we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion,survival,and proliferation. We show that iPSC lines generated using Oct-3/4,Sox-2,Nanog,and Lin-28 express a repertoire of integrins similar to that of hESCs,with prominent expression of subunits alpha5,alpha6,alphav,beta1,and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin,in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel,as shown by immunological blockade experiments. On vitronectin,the integrin alphavbeta5 was required for initial attachment,but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore,iPSCs cultured on vitronectin for 9 passages retained normal karyotype,pluripotency marker expression,and capacity to differentiate in vitro. These studies suggest that vitronectin,or derivatives thereof,might substitute for Matrigel in a more defined system for iPSC culture. 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

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).backslashnbackslashnMETHODS AND RESULTS: To induce endothelial cell differentiation,undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days,CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation,these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%),CD31 (97.2+/-2.5%),and VE-cadherin (93.7+/-2.8%),form vascular-like channels,and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward,5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly,bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally,in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).backslashnbackslashnCONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs,and enhance their angiogenic effects in vivo. Furthermore,activation of nAChRs has anti-apoptotic,angiogenic,and proliferative effects through MAPK and Akt signaling pathways. View Publication

Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this,we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness,many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells. View Publication

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However,traces of potentially oncogenic genes remaining in actively transcribed regions of the genome,limit their potential for use in human therapeutic applications. Additionally,non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context. In this video,we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR),which has an advantage over less sensitive techniques previously used to detect gene expression differences. Full conversion into clinical-grade good manufacturing practice (GMP) conditions,allows human clinical relevance. Our protocol offers another methodology--provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications--for deriving GMP-grade hiPSCs,which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions. View Publication

Extracellular vesicles (EVs) are phospholipid-based particles endogenously produced by cells. Their natural composition and selective cell interactions make them promising drug carriers. However,in order to harness their properties,efficient exogenous drug encapsulation methods need to be investigated. Here,EVs from various cellular origins (endothelial,cancer and stem cells) were produced and characterised for size and composition. Porphyrins of different hydrophobicities were employed as model drugs and encapsulated into EVs using various passive and active methods (electroporation,saponin,extrusion and dialysis). Hydrophobic compounds loaded very efficiently into EVs and at significantly higher amounts than into standard liposomes composed of phosphocholine and cholesterol using passive incubation. Moreover,loading into EVs significantly increased the cellular uptake by textgreater60% and the photodynamic effect of hydrophobic porphyrins in vitro compared to free or liposome encapsulated drug. The active encapsulation techniques,with the saponin-assisted method in particular,allowed an up to 11 fold higher drug loading of hydrophilic porphyrins compared to passive methods. EVs loaded with hydrophilic porphyrins induced a stronger phototoxic effect than free drug in a cancer cell model. Our findings create a firm basis for the development of EVs as smart drug carriers based on straightforward and transferable methods. View Publication

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells,we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture,which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However,we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture,although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand,all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells,our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation. View Publication

Since the derivation of human embryonic stem (hES) cells,their translation to clinical therapies has been met with several challenges,including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We,along with other groups,have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence,flow cytometry,and teratoma formation assays,we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly,the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices,which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies. View Publication