技术资料

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

Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations,convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors,measuring yield and purity outputs of undifferentiated,neuroectoderm,mesendoderm,and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically,small interfering RNA knockdown of RAPTOR,a component of mTOR complex 1,phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold,with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives. 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

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling,and differentiation is promoted by BMP signaling,but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium,we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG,OCT4,and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal. View Publication

BACKGROUND The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases,the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. METHODS AND RESULTS Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug,which elicits lethargy,anorexia,weight loss,and peritoneal fibrosis,all of which confound the interpretation of autophagy. Given this,we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms,reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation,we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely,animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. CONCLUSIONS Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. 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

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

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. 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

BACKGROUND Neuritic degeneration is an important early pathological step in neurodegeneration. AIM The purpose of this study was to explore the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of endoplasmic reticulum (ER) and mitochondria. METHODS Here,we set up neuritic degeneration models by neurite cutting-induced neural degeneration in human-induced pluripotent stem cell-derived neurons. RESULTS We found that neuritic ER becomes fragmented and forms complexes with mitochondria,which induces IP3R-dependent mitochondrial Ca(2+) elevation and dysfunction during neuritic degeneration. Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca(2+) elevation during neuritic degeneration. Mechanically,tightening of the ER-mitochondria associations by expression of a short synthetic linker" and ER Ca(2+) releasing together could promote mitochondrial Ca(2+) elevation� View Publication

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here,for the first time,human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells,exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture,the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs,suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However,continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells,linking embryonic genotoxic exposure to genomic instability. View Publication