技术资料

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units,and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture,the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality. View Publication

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According to the current model of adult hematopoiesis,differentiation of pluripotential hematopoietic stem cells into common myeloid- and lymphoid-committed progenitors establishes an early separation between the myeloid and lymphoid lineages. This report describes a rare and previously unidentified CD45R-CD19+ B cell progenitor population in postnatal bone marrow that can also generate macrophages. In addition to the definition of this B-lineage intermediate,the data indicate that a developmental relationship between the B and macrophage lineages is retained during postnatal hematopoiesis. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

Summary Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290,which causes missplicing and premature termination,but the basis of this sensitivity is unclear. Here,we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups,despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups,explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290,restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention. View Publication

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic,plastic-adherent,self-renewing cells that are capable of tri-lineage differentiation into bone,cartilage or fat in vitro. Thus,MSCs are promising candidates for cell-based medicine. However,classifications of MSCs have been defined retrospectively; moreover,this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1,and this population may be analogous to murine PDGFR??+Sca-1+ cells,which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence,which provided definitive proof of neural crest lineage,however,technical considerations prevent the use of a similar approach to determine the origin of human LNGFR+THY-1+ MSCs. To further clarify the origin of human MSCs,human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells,human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR+THY-1+ neural crest-like cells,designated as LT-NCLCs,showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore,we transplanted LT-NCLCs into chick embryos,and traced their potential for survival,migration and differentiation in the host environment. These results suggest that LNGFR+THY-1+ cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs. View Publication

Acute monocytic leukemia (AML-M5),a subtype of acute myeloid leukemia (AML),affects mostly young children and has poor prognosis. The mechanisms of treatment failure of AML-M5 are still unclear. In this study,we generated iPSC from THP-1 cells from a patient with AML-M5,using retroviruses encoding the pluripotency-associated genes (OCT3/4,SOX2,KLF4 and c-MYC). These AML-M5-derived iPSC showed features similar with those of human embryonic stem cells in terms of the morphology,gene expression,protein/antigen expression and differentiation capability. Parental-specific markers were down-regulated in these AML-M5-derived iPSCs. Expression of MLL-AF9 fusion gene (previously identified to be associated with pathogenesis of AML-M5) was observed in all iPSC clones as well as parental cells. We conclude that AML-M5-specific iPSC clones have been successfully developed. This disease model may provide a novel approach for future study of pathogenesis and therapeutic intervention of AML-M5. View Publication

The unique mental abilities of humans are rooted in the immensely expanded and folded neocortex,which reflects the expansion of neural progenitors,especially basal progenitors including basal radial glia (bRGs) and intermediate progenitor cells (IPCs). We found that constitutively active Sonic hedgehog (Shh) signaling expanded bRGs and IPCs and induced folding in the otherwise smooth mouse neocortex,whereas the loss of Shh signaling decreased the number of bRGs and IPCs and the size of the neocortex. SHH signaling was strongly active in the human fetal neocortex but Shh signaling was not strongly active in the mouse embryonic neocortex,and blocking SHH signaling in human cerebral organoids decreased the number of bRGs. Mechanistically,Shh signaling increased the initial generation and self-renewal of bRGs and IPC proliferation in mice and the initial generation of bRGs in human cerebral organoids. Thus,robust SHH signaling in the human fetal neocortex may contribute to bRG and IPC expansion and neocortical growth and folding. View Publication

Loss-of-function studies are fundamental for dissecting gene function. Yet,methods to rapidly and effectively perturb genes in mammalian cells,and particularly in stem cells,are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways,and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid,reversible,titratable and independent manner. By engineering molecular tuners for NANOG,CHK1,p53 and NOTCH1 in mammalian stem cells,we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes. View Publication