技术资料

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs,the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8),which are expressed in simple epithelial cells,while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Human embryonic stem cells (hESCs) require specific niches for adhesion,expansion,and lineage-specific differentiation. In this study,we showed that a membrane substrate offers better tissue niches for hESC attachment,spreading,proliferation,and differentiation. The cell doubling time was shortened from 46.3±5.7 h for hESCs grown on solid substrates to 25.6±2.6 h for those on polyester (PE) membrane substrates with pore size of 0.4 μm. In addition,we observed an increase of approximately five- to ninefold of definitive endoderm marker gene expression in hESCs differentiated on PE or polyethylene terephthalate membrane substrates. Global gene expression analysis revealed upregulated expressions of a number of extracellular matrix and cell adhesion molecules in hESCs grown on membrane substrates. Further,an enhanced nuclear translocation of β-catenin was detected in these cells. These observations suggested the augmentation of Wnt signaling in hESCs grown on membrane substrates. These results also demonstrated that a membrane substrate can offer better physicochemical cues for enhancing in vitro hESC attachment,proliferation,and differentiation. View Publication

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation,physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation,cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. View Publication

The glycogen synthase kinase 3 (GSK3) inhibitor,6-bromoindirubin-3'-oxime (BIO),is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However,the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore,its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures,as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies,as well as expression of pluripotent markers,Oct4,Nanog,Sox2,C-myc,Klf4,E-cadherin,and the proliferative markers,Pcna and C-myc. In contrast,there was no significant change in expression of apoptosis markers,P53,P21 and cyclin-related genes (Cyclin A,CDK2,Cyclin D1),as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO,EBs were formed,which were capable of further differentiating into various cell types found in the three embryonic germ layers,as determined by immunofluorescence and/or histologic staining. In conclusion,adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state. View Publication

In this unit,we describe a simple coculture-free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow-derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured,the difficulty in introducing specific genetic changes into these cells,and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cells from embryonic stem cells addresses these limitations. ES-derived mast cells can be generated in numbers sufficient for studies of the pathways involved in mast cell effector functions. These ES cell-derived mast cells respond to antigens and other stimuli by releasing histamine,cytokines,lipids,and other bioactive mediators. The derivation of human mast cells from ES cells carrying mutations introduced by homologous recombination should provide a novel means of testing the function of genes in both the development and the effector functions of mast cells. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells,and is responsible for the radioactive iodine accumulation. However,treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression,(125)I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells,as was previously done for mouse embryonic stem cells. First,we obtained definitive endoderm from human ESCs. Second,we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors,with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency,endoderm and thyroid markers and (125)I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood. View Publication

Embryonic and induced pluripotent stem cells (IPSCs) derived from mammalian species are valuable tools for modeling human disease,including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. Here we used transgenic common marmoset (Callithrix jacchus) and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP) reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures,we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications. View Publication

Cellular reprogramming from adult somatic cells into an embryonic cell-like state,termed induced pluripotency,has been achieved in several cell types. However,the ability to reprogram human amniotic epithelial cells (hAECs),an abundant cell source derived from discarded placental tissue,has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs),but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore,AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation,including NEUROD1 and SOX17,markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs,we analyzed global DNA methylation,global histone acetylation,and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts,hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise,quantitative gene expression analyses show that hAECs endogenously express OCT4,SOX2,KLF4,and c-MYC,all four factors used in cellular reprogramming. Thus,hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. View Publication

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine because they can undergo unlimited self-renewal and retain the capability to differentiate into all cell types in the body. Although numerous genes/proteins such as Oct4 and Gata6 have been identified to play critical regulatory roles in self-renewal and differentiation of hESC,the majority of the regulators in these cellular processes and more importantly how these regulators co-operate with each other and/or with epigenetic modifications are still largely unknown. We propose here a systematic approach to integrate genomic and epigenomic data for identification of direct regulatory interactions. This approach allows reconstruction of cell-type-specific transcription networks in embryonic stem cells (ESCs) and fibroblasts at an unprecedented scale. Many links in the reconstructed networks coincide with known regulatory interactions or literature evidence. Systems-level analyses of these networks not only uncover novel regulators for pluripotency and differentiation,but also reveal extensive interplays between transcription factor binding and epigenetic modifications. Especially,we observed poised enhancers characterized by both active (H3K4me1) and repressive (H3K27me3) histone marks that contain enriched Oct4- and Suz12-binding sites. The success of such a systems biology approach is further supported by experimental validation of the predicted interactions. View Publication