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

Estrogen (E2) deficiency is responsible for increased bone turnover in the postmenopausal period,and it can be prevented by estrogen replacement therapy. The way estrogen acts on bone cells is not fully understood. Human bone marrow cell cultures may be a reliable model for studying the action of steroids on osteoclastogenesis in vitro. We examine the effects of estradiol and Raloxifene,a selective estrogen receptor modulator,on human primary bone marrow cells cultured for 15 days. 17beta-estradiol and Raloxifene significantly decreased the number of tartrate-resistant acid phosphatase multinucleate cells from osteoclast precursors on day 15. Estrogen receptor alpha (ER-alpha) mRNA was present in bone marrow mononuclear cells cultured for 5 days,but there was no estrogen receptor beta (ER-beta) mRNA,suggesting that this effect was mediated by ER-alpha. 15-day cultures no longer contained ER-alpha mRNA,suggesting that estrogen acts on early events of osteoclast differentiation. Finally,10-8 M 17beta-estradiol has no effect on the release of IL-6 and IL-6-sr into the medium of marrow mononuclear cells cultured for 5 or 15 days. Osteoclast apoptosis was not affected by estradiol or Raloxifene after 15 days of culture under our conditions. In conclusion,we have shown that both estradiol and Raloxifene inhibit osteoclast differentiation in human bone marrow mononuclear cultures. The biological effect that can mimic in vivo differentiation could be mediated through ER-alpha. 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

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism,the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity,osteocalcin production,and mRNA levels for alkaline phosphatase,type I alpha 2-procollagen,and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant,but varied with the incubation conditions. At high cell density and in the presence of serum,TGF beta decreased alkaline phosphatase activity. However,at low cell density and under serum-free conditions,TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%,while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation,mRNA levels were measured at 24 hours. Individually,TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen,but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels,but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors. View Publication

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

To directly study the biological properties of purified hematopoietic colony-forming cell precursors,cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting,and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3),IL-6,erythropoietin,and mast cell growth factor (a c-kit ligand),cell numbers increased approximately 10(6)-fold over a period of 4 wk,and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly,the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured,extensive cell production was repeated,again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool. View Publication

Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs,the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here,we describe a method based on the use of a small-molecule GSK3?? inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3?? inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover,we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free,efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies,in vitro screenings,and disease modeling. ?? 2013 The Authors. View Publication

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However,the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH(Bright) (ALDH(Br)) cell subsets for their phenotype and proliferative capability. In this study,we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin(-)) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin(-)CD34(+)CD38(Low/-) cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH(Br) cells when associated with SP functionality (SP/ALDH(Br) fraction). Furthermore,the SP marker identified G(0) cells in all ALDH fractions,allowing us to sort quiescent cells regardless of ALDH activity. Moreover,we show that,within the Lin(-)CD34(+)CD38(-)ALDH(Br) population,the SP marker identifies cells with higher primitive characteristics,in terms of stemness-related gene expression and in vitro and in vivo proliferative potential,than the Lin(-)CD34(+) CD38(-)ALDH(Br) main population cells. In conclusion,our study shows that the coexpression of SP and ALDH markers refines the Lin(-)CD34(+)CD38(-) hematopoietic compartment and identifies an SP/ALDH(Br) cell subset enriched in quiescent primitive HSCs/HPCs. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly,silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1,whereas adhesion to VCAM-1 was reduced. Thus,PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells,whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4. View Publication

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency,self-derivation and potential ability to differentiate into motoneurons. However,it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo,which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study,we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system,human iPSCs efficiently differentiated into motoneurons which displayed typical morphology,expressed specific molecules,and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats,they survived robustly,extended axons along the nerve,and formed functional connections with the target muscle (biceps brachii),thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo,and they have potential for stem cell-based therapies for motoneuron diseases. textcopyright 2013 Elsevier B.V. View Publication

The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here,we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B,two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3,a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680,which has recently entered phase II clinical trials for cancer treatment,reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus,although our studies raise serious doubts on the application of reversine in regenerative medicine,they support the paradigm that reversine might be a useful agent in cancer chemotherapy. View Publication