技术资料

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

Because video data are complex and are comprised of many images,mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article,we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments,hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies,recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth,three recipes were created. The first segmented the image into the colony and background,the second enhanced the image to define colonies throughout the video sequence accurately,and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes,the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared,results were virtually identical,indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition,other video bioinformatics recipes can be developed in the future for other cell processes such as migration,apoptosis,and cell adhesion. View Publication

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function,little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages,to migrate toward stromal cell-derived factor 1α,and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However,the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast,adhesion to the extracellular matrix was inhibited by silencing of talin-1,but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity,independent of integrin functions. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However,the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound,vitamin C (Vc),enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence,a recently identified roadblock for reprogramming. In addition,Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker,TBR2,and also glial marker,S100β. In contrast,inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers,PAX6 and EAAT1,respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. View Publication

Chromatin regulation is critical for differentiation and disease. However,features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches,we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably,we found that the chromatin environment of Ewing sarcoma,a mesenchymally derived tumor,is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements,a feature associated with differentiation and oncogenesis. View Publication

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However,development of cell-based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII mice,we characterized the distribution of lineage-depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDH(hi) or ALDH(hi)CD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues,including islet and ductal regions of mouse pancreata. In contrast,variable phenotypes were detected in the chimeric liver,with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45-/HLA- cells with diluted GUSB expression predominant in the liver parenchyma. However,true nonhematopoietic human (HLA+/CD45-) cells were rarely detected in other peripheral tissues,suggesting that these GUSB+/HLA-/CD45- cells in the liver were a result of downregulated human surface marker expression in vivo,not widespread seeding of nonhematopoietic cells. However,relying solely on continued expression of cell surface markers,as used in traditional xenotransplantation models,may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes,indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. View Publication

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected,we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs,where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection,as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability,expansion,and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro,it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether,these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression. View Publication

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here,we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes,respectively. Clusters of Wnt targets showed enrichment in specific biological functions,such as gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover View Publication

Human hematopoietic stem cells are defined by their ability to repopulate multiple hematopoietic lineages in the bone marrow of transplanted recipients and therefore are functionally distinct from hematopoietic progenitors detected in vitro. Although factors capable of regulating progenitors are well established,in vivo regulators of hematopoietic repopulating function are unknown. By using a member of the vertebrate Wnt family,Wnt-5A,the proliferation and differentiation of progenitors cocultured on stromal cells transduced with Wnt-5A or treated with Wnt-5A conditioned medium (CM) was unaffected. However,i.p. injection of Wnt-5A CM into mice engrafted with human repopulating cells increased multilineage reconstitution by textgreater3-fold compared with controls. Furthermore,in vivo treatment of human repopulating cells with Wnt-5A CM produced a greater proportion of phenotypically primitive hematopoietic progeny that could be isolated and shown to possess enhanced progenitor function independent of continued Wnt-5A treatment. Our study demonstrates that Wnt-5A augments primitive hematopoietic development in vivo and represents an in vivo regulator of hematopoietic stem cell function in the human. Based on these findings,we suggest a potential role for activation of Wnt signaling in managing patients exhibiting poor hematopoietic recovery shortly after stem cell transplantation. View Publication

Despite the global prevalence of gastric disease,there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium,and that β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types,including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology,and also represent a new platform for drug discovery. View Publication