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

NUP98-HOXA9,the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation,is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation,proliferation,and gene expression in primary human CD34+ hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture,cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation,suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term culture-initiating cells,the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation,oligonucleotide microarray analysis was done at several time points over 16 days,starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNbeta1 and accompanied by marked up-regulation of IFN-induced genes,peaking at 3 days posttransduction. In contrast,oncogenes such as homeobox transcription factors,FLT3,KIT,and WT1 peaked at 8 days or beyond,coinciding with increased proliferation. In addition,several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation,differentiation,and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9. View Publication

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis,induced by injection of the TLR-2 ligand,zymosan,we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein,we show that only in low-dose injected mice,the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose,the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However,the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity,i.e.,CD34 and AC133. In addition to differences in surface marker expression,the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast,the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile,the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells. View Publication

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here,we show that SB-216763,a glycogen synthase kinase-3 (GSK3) inhibitor,can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4,Sox2,and Nanog. Furthermore,Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers,and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge,SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months. View Publication

Thymosin $\beta$4 (T$\beta$4) is a G-actin sequestering protein that contributes to diverse cellular activities,such as migration and angiogenesis. In this study,the beneficial effects of combined cell therapy with T$\beta$4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with T$\beta$4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly,T$\beta$4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with T$\beta$4 significantly increased cell migration and sprouting from microbeads. Moreover,additional treatment with T$\beta$4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the T$\beta$4-hASCs combination on vessel recruitment,dorsal window chambers were transplanted,and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with T$\beta$4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together,the therapeutic application of hASCs combined with T$\beta$4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia. View Publication

In this report,we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for textgreater or = 7 days was found to irreversibly terminate CFU-pre-B production and,in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in textgreater 70% of cultures 1 week after the switch),the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells,regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction,as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC,thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro,without the need for their prior purification,should facilitate studies of totipotent hematopoietic stem cell regulation. View Publication

Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells. View Publication

Production of new neurons throughout adulthood has been well characterized in two brain regions,the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ (Bennett et al.). The present study demonstrates that NSCs derived from two astrogliogenic CVOs,the median eminence and organum vasculosum of the lamina terminalis of the nestin-GFP mouse,possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs,following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice,migrate caudally to the SVZ and express early neuronal markers (TUC-4,PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU(+) cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively,these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ,they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain. View Publication

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood,as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow,detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al,2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens,with particular attention to the expression of ALDH on erythroid precursors. To this aim,we used three kinds of approach: i) multidimensional analytical flow cytometry,detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells,followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow,we identified three populations of cells,namely ALDH+CD34+,ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52,0.53 and 0.57,respectively). As compared to ALDH-CD34+ cells,ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells,with brighter expression of CD117 and CD133,accompanied by lower display of CD38 and CD45RA. Of interest,ALDH+CD34- population disclosed a straightforward erythroid commitment,on the basis of three orders of evidences. First of all,ALDH+CD34- cells showed a CD71bright,CD105+,CD45- phenotype. Secondly,induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally,ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study,comparing surface antigen expression of ALDH+/CD34+,ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow,clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. View Publication

BackgroundOP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However,the whole co-culture procedure usually needs 14–18 days,including preparing OP9 cells at least 4 days. Therefore,the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.MethodsIn the experimental group,we set six different densities of OP9 cells and just cultured them for 24 h before co-culture,and in the control group,OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them.ResultsOP9 cells were randomly assigned into two groups. In the experimental group,six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast,in the control group,OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining,and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day,followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group.ConclusionThe peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency. View Publication

BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments." View Publication