参考文献

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

BACKGROUND: Mesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However,in most models,MSC were added at the same time of lymphocyte stimulation,which is quite different from what occurs in vivo. AIMS: To investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Wharton's jelly stromal cells (WJSC). METHODS: WJSC,licensed or not with activation-related cytokines,were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture. RESULTS: Lymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines,in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan,suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect,in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h,and added afterwards to non-licensed WJSC,showed normal or even increased proliferation. On the other hand,their proliferation was strongly inhibited by licensed WJSC. CONCLUSIONS: WJSC have a potent immunosuppressive function best realized with direct contact,and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders. View Publication

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs,the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus,the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However,MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation,we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore,umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs. View Publication

Embryonic stem cells,derived from the inner cell mass of murine blastocysts,can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes,such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta,during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes. View Publication

Adult hepatocytes are polarised with their apical and basolateral membranes separated from neighbouring cells by tight junction proteins. Although efficient differentiation of pluripotent stem cells to hepatocytes has been achieved,the formation of proper polarisation in these cells has not been thoroughly investigated. In the present study,human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs) were differentiated to hepatocyte-like cells and the derived hepatocytes were characterised for mature hepatocyte markers. The secretion of hepatic proteins,expression of hepatic genes and the functional hepatic polarisation of stem cell-derived hepatocytes,foetal hepatocytes and the HepG2 hepatic cell line were evaluated and the different lines were compared. The results indicate that hESC-derived hepatocytes are phenotypically more robust and functionally more efficient compared with the hMSC-derived hepatocytes,suggesting their suitability for toxicity studies. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However,current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation,which is required to generate endodermal,ectodermal,and mesodermal cell lineages. We report a new,straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs,which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1,GAG,and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats,magnetic resonance imaging studies showed gradual engraftment,and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs,which could improve cartilage regeneration outcomes in arthritic joints. View Publication

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However,tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs,especially those in cancer patients. To circumvent these issues,we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable,but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition,invasion,stemness,and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ,downstream protumor factors,and hyaluronan and its cofactor TSG6,which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf" therapies and bioengineering applications." View Publication

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton,cornea,peripheral nervous system,and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),further modifications are required to improve the robustness,efficacy,and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions,the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons,glia,melanocytes,and corneal endothelial cells. In addition,cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine. View Publication

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids,rather than two-dimensional (2D) monolayers,is advantageous for many regenerative,biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly,we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells,where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. View Publication

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically,various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods,we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods,which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming,thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication