技术资料

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental. View Publication

Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development,however,must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor,EphB4,known to be spatially restricted in expression and critical for organized vessel formation,modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast,blood cell,cardiomyocyte,and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated,but not neuroectoderm-associated,genes. Therefore,EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization. View Publication

In this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC)-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts,patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina,ciliary margin,and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture,the retinal organoids autonomously generated stratified retinal tissues,including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation,has been validated in two lines of human pluripotent stem cells,and provides insight into optic cup invagination in vivo. View Publication

Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined,xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free,single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 $$M Rho-associated kinase inhibitor Y-27632 into two types of xeno-free,defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (˜90 %) and cell expansion (˜70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells. View Publication

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings,however,requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study,we show that once specified to neuro-epithelial lineage,hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However,our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes,namely,migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes,a new potential source for therapeutic use for brain tumors and other neurological diseases. View Publication

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms,yet their clinical translation has been compromised by their biosafety concern. Here,we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/ progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immu-nodeficient mice. Moreover,removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplanta-tion,ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable,depending on the oncogenic load,with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus,transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:694–702 SIGNIFICANCE Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products,especially when reprogrammed with integrating vectors. Two major under-lying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzy-matic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in test-ing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature b cell phenotype would lead to safe islet replacement therapy for diabetes. View Publication

Human embryonic stem cell (hESC) line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation,c.3536delA,of FBN1 gene. Characteristic tests proved that the hESC line presented typicalmarkers of pluripotency and had the capability to formthe three germlayers both in vitro and in vivo. View Publication

We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC),which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore,and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines,but never in stem cells,thus limiting their potential therapeutic application. In this work,we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency,which were stably maintained without selection for 3 months. Importantly,no integration of the HAC DNA was observed in the hESc lines,compared with the fibrosarcoma-derived control cells,where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency,differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc,and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. View Publication

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform,combined with image cytometry,enables the systematic analysis of multiple simultaneously detected marker expression in individual cells,for screening of various chemically defined media across hPSC lines,and the study of phenotypic responses. View Publication

Under defined differentiation conditions,human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate,the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening,a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification,and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2,increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus,we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. View Publication

Pluripotent stem cells provide an invaluable tool for generating human,disease-relevant cells. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system,characterized by myelin damage. Oligodendrocytes are the myelinating cells of the central nervous system (CNS); they differentiate from progenitor cells,and their membranes ensheath axons,providing trophic support and allowing fast conduction velocity. The current understanding of oligodendrocyte biology was founded by rodent studies,where the establishment of repressive epigenetic marks on histone proteins,followed by activation of myelin genes,leads to lineage progression. To assess whether this epigenetic regulation is conserved across species,we differentiated human embryonic and induced pluripotent stem cells to oligodendrocytes and asked whether similar histone marks and relative enzymatic activities could be detected. The transcriptional levels of enzymes responsible for methylation and acetylation of histone marks were analyzed during oligodendrocyte differentiation,and the post-translational modifications on histones were detected using immunofluorescence. These studies showed that also in human cells,differentiation along the oligodendrocyte lineage is characterized by the acquisition of multiple repressive histone marks,including deacetylation of lysine residues on histone H3 and trimethylation of residues K9 and K27. These data suggest that the epigenetic modulation of oligodendrocyte identity is highly conserved across species. View Publication