搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here,we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition,SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore,through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether,our results indicate that even an extremely homogeneous HSC population,based on phenotype and dye efflux,cannot reconstitute mice at absolute efficiencies. View Publication

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. View Publication

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGF? signaling (A83-01) and FGF signaling (PD173074),called BAP,can efficiently differentiate to extraembryonic (ExE) cells in vitro. Due to restricted access to human embryos,it is ethically impossible to test the developmental potential of ExE cells in vivo. Here,we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation,ExE cells contributed to the mouse placenta. More interestingly,ExE cells could chimerize with the mouse blastocyst as,after injection into the blastocyst,they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice,human cells were found at E14 in placental labyrinth,junction zones,and even near the uterine decidua,expressed placental markers,and secreted human chorionic gonadotropin. Surprisingly,ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9,consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2,a mesodermal determinant,restricted the contribution of ExE cells to the placenta. Thus,we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells. View Publication

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. View Publication

Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient,strain-dependent,and requires expert skills. Over recent years,several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment,which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin,which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary,our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%. View Publication

Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term,multilineage reconstitution of hematopoiesis in recipients,with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however,variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary,despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype,uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ. View Publication

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation,1.21% of transplanted hNPCs survived. In these hNPCs,parvalbumin (PV)-,calretinin (CR)-,somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-,CR-,and SS-positive cells among GFP+ cells were 35.5%,15.7%,and 17.1%,respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude,frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion,GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy. View Publication