技术资料

Down's syndrome is a common disorder with enormous medical and social costs,caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene,XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases,we inserted a large,inducible XIST transgene into the DYRK1A locus on chromosome 21,in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications,chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21,free from genetic and epigenetic noise. Notably,deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy'. View Publication

Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis,and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial,cardiac,and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here,we describe a rapid and robust NC differentiation method called LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily� View Publication

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication

PURPOSE: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).backslashnbackslashnMETHODS: Sixty-nine rats (38 male,31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1,6,and 12 months after surgery. Both ocular tissues and systemic organs (brain,liver,kidneys,spleen,heart,and lungs) were fixed in 4% paraformaldehyde,embedded in paraffin,and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker),anti-TRA-1-85 (human cell marker),anti-Ki67 (proliferation marker),anti-CD68 (macrophage),and anti-cytokeratin (epithelial marker).backslashnbackslashnRESULTS: The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P textless 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.backslashnbackslashnCONCLUSIONS: hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. View Publication

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication

Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi),as well as constitutive heterochromatin,replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs),the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome- specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus,the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However,the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory. View Publication

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy,drug screening and toxicity testing of neural degenerative diseases. However,the molecular regulation of their proliferation and apoptosis,which needs to be revealed before clinical application,is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here,we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells,hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently,global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically,a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression,revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover,forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly,we found that paraquat,a neurotoxin,not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs,indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably,inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively,miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. View Publication

Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse,non-primate human,and human; however,the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles,feeder cell populations,such as mouse embryonic fibroblasts (MEFs),are required to stimulate growth and development. Hence,in this report,we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival,growth,and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as $$MEM/F12 supplemented with fetuin,insulin,transferrin,selenium,and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles,which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore,80% of the oocytes from surviving follicles with an initial diameter of 90-100 µm underwent germinal vesicle breakdown (GVBD),and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture,yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation. View Publication

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes,but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However,the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore,we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo.backslashnbackslashnMETHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte,Laguna Hills,CA,USA) devices into diabetic mice.backslashnbackslashnRESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing textgreater80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices,despite lacking direct contact with the host environment,and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells.backslashnbackslashnCONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device,yielding glucose-responsive insulin-producing cells capable of reversing diabetes. View Publication

Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome,slow,and variable. Alternatively,human fibroblasts can be directly converted into induced neuronal (iN) cells. However,with present techniques conversion is inefficient,synapse formation is limited,and only small amounts of neurons can be generated. Here,we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin,form mature pre- and postsynaptic specializations,and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples,our approach enables large-scale studies of human neurons for questions such as analyses of human diseases,examination of human-specific genes,and drug screening View Publication

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However,fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs,and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs,allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs. View Publication

We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium,hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium,possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further,these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful,objective,measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny,particularly changes induced by the cellular environment. View Publication