技术资料

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

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

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

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature,with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries,including planar 2-D films,macroporous 3-D sponges,and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However,synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion,viability,proliferation,self-renewal,and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover,the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment,where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus,synthetic substrates with specific 3-D microgeometries can support hESC colony formation,self-renewal,and directed differentiation to multiple lineages while obviating the stringent needs for complex,exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

Embryonic stem cells (ESCs) are unique cells,which have the ability to differentiate into all cell types that comprise the adult organism. Furthermore,ESCs can infinitely self-renew under optimized conditions. These features place human ESCs (hESCs) in a position where these cells can be exploited for tissue engineering and regenerative medicine approaches in treating human degenerative disorders. However,cell therapy approaches will require large amounts of clinically useable cells,not typically achievable using standard static cell culture methods. Here,we describe a method wherein clinically relevant numbers of hESCs can be generated in a cost and time effective manner. 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

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this,time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis,including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique,in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work,and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality,fast chromosomal analysis of human ES and iPS cells. View Publication

More than 10 years after their first isolation,human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable,and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever,biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper,we review the cutting edge of this exciting area of translational research. View Publication

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH),a DA neuron marker,and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth. View Publication

In guiding hES cell technology toward the clinic,one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So,we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture,including the developmental potential to differentiate into representative tissues of all three embryonic germ layers,unlimited and undifferentiated proliferative ability,and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF,insulin-like growth factor 2 (IGF-2),and transforming growth factor β (TGF-β),thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together,bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically. View Publication

BACKGROUND: The radiation-induced bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However� View Publication