技术资料

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years,evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review,we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition,we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Human induced pluripotent stem cells (hiPSCs),like embryonic stem cells,are under intense investigation for novel approaches to model disease and for regenerative therapies. Here,we describe the derivation and characterization of hiPSCs from a variety of sources and show that,irrespective of origin or method of reprogramming,hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium,CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage,we were able to demonstrate by single cell analysis (flow cytometry),that hiPSC-derived erythroblasts express alpha globin as previously described,and that a sub-population of these erythroblasts also express haemoglobin F (HbF),indicative of fetal definitive erythropoiesis. More notably however,we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner,but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover,the HbA expressing erythroblast population could be greatly enhanced (44textperiodcentered0 ± 6textperiodcentered04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and human-induced pluripotent stem cells,are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes,large numbers of high-quality cells are essential. Recently,we showed that a biological substrate,recombinant E8 fragments of laminin isoforms,sustains long-term self-renewal of hPSCs in defined,xeno-free medium with dissociated single-cell passaging. Here,we describe a modified culture system with similar performance to efficiently expand hPSCs under defined,xeno-free conditions using a non-biological synthetic substrate. View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials,high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end,we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous,homogenous process.Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83. ??. 2.56%; myosin heavy chain (MHC): 19.30. ??. 5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50. ??. 7.35%; MHC: 42.85. ??. 2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3. days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT. textless. 5%; MHC. textless. 2%). Simply by applying intermittent agitation during these 3. days followed by continuous agitation for the subsequent 9. days,CM differentiation efficiency can be substantially increased (cTNT: 65.73. ??. 10.73%; MHC: 59.73. ??. 9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control.During the hESC expansion phase,cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166??88??105??m2) achieving a cell concentration of 3.74??0.55??106cells/mL (18.89??2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively,the integrated MC rocker platform produced 190.5??58.8??106 CMs per run (31.75??9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines,hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug,Isoproterenol on beating frequency.In conclusion,we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. ?? 2014. View Publication

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood,aside from numerous congenital abnormalities. FA mouse models have been generated; however,they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero,a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology,we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential,disruption of the second allele causes a severe growth disadvantage. As a result,heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele,quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning,this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. ?? 2014. View Publication

Using time-lapse imaging,we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating,and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore,the daughter cells showed a continued pattern of cell death after division,so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact,which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast,most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny,without the need for cell:cell contacts and independent of their motility patterns. ?? 2014 The Authors. View Publication

Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell,showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however,its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation,induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells,we hypothesize that it will induce alterations in morphology,inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology,viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology,decrease of cell viability,and induction of genotoxicity in IPS. View Publication

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors,murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However,MLV vectors have been implicated in malignancy induced by insertional mutagenesis,whereas lentiviral vectors have not. Furthermore,the infectivity of MLV vectors is limited to dividing cells,whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells,allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone,thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state,and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy,we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5),the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression,we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines. 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