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

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

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs,although its contribution to stem cell fate remains unknown. In this study,we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover,short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells,the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells,compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist,all-trans-retinoic acid,suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs,suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs. View Publication

We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained,significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow,or to alpha4-deficient endothelial cells or pericytes,we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells,suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells,the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant,ongoing signaling required for bone marrow retention of progenitor cells during homeostasis. View Publication

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study,we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation,the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry,immunoblotting,and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore,the cells were tested for functions associated with mature hepatocytes,including glycogen storage and indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate,numerous epithelial cells resembling hepatocytes were observed,and approximately 74% of the cells expressed the hepatic marker,albumin,after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers,and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development,and could provide a source of hepatocytes for transplantation therapy and drug screening. View Publication

Diffuse alveolar hemorrhage (DAH),although rare,is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans,although increasingly neutrophils,NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model,we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress,namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model,with accompanying reduction in IFN-driven genes and pathology. Lastly,coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH. View Publication

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However,the current conventional method leads to poor recovery of hPSCs after thawing. Here,we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing,we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival,whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover,the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. View Publication

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However,currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability,painful and invasive cell-harvesting procedures,and tumorigenesis. Previously,we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan,fibromodulin (FMOD),circumventing gene transduction. In this study,we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect,contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence,engraftment,and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together,we have provided an extended potency,safety,and molecular profile of FReP cell-based bone regeneration. Therefore,FReP cells present a high potential for cellular and gene therapy products for bone regeneration. View Publication