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

[3H]thymidine uptake by NFS-60 cells in microcultures was found to increase in a linear fashion with the increasing doses of purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). Such increases were found neither with rhG-CSF samples pretreated with rabbit anti-rhG-CSF serum nor with other human colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (hGM-CSF) or macrophage colony-stimulating factor (hM-CSF). Based on these findings,sera from normal persons and patients with severe infections or various hematological disorders were tested after dialysis using this system in order to determine whether G-CSF levels in sera can be estimated or not. In ten normal persons,five patients with acute myelogenous leukemia (AML M1,M2,and M3),five with myelodysplastic syndrome,and four with chronic myelogenous leukemia,no increases in [3H]thymidine uptake were found within the dose range of 0.4 microliters to 50 microliters. In contrast,linear dose responses parallel to a G-CSF standard curve were observed in one patient with a severe bacterial infection,four with aplastic anemia,two with acute myelomonocytic leukemia (AMMoL) (M4),and two with idiopathic neutropenia tested. From the standard curve,the probable levels of G-CSF were calculated as follows: approximately 200 pg/ml with infection,130-220 pg/ml with aplastic anemia,150 and 200 pg/ml with AMMoL,and 1120 and 1200 pg/ml with idiopathic neutropenia. The activities of sera were reduced by the anti-rhG-CSF serum pretreatment in the same way as documented in the case of rhG-CSF. Furthermore,the level in a patient with a severe infection became undetectable soon after elimination of the infection and blood neutrophil counts had returned to normal. These findings indicate that the microbioassay system will be useful for measuring circulating G-CSF levels which would fluctuate in accord with requirements for stimulating neutrophil production or with abnormal production of hG-CSF. View Publication

Human embryonic stem cells (hESCs) are used as platforms for disease study,drug screening and cell-based therapy. To facilitate these applications,it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However,the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However,certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site,probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein,LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. View Publication

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors,we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells,suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies,under the influence of Stat5 signaling,a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing,mainly myeloid,differentiation. When these cells are injected into lethally irradiated mice,they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal,and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells. View Publication

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts,an effect achieved either alone or in combination with differentiating agents,such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore,valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4,a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary,we here show that valproic acid,known to induce differentiation or apoptosis in leukemic blasts,stimulates the proliferation of normal HSC,an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH),hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon,we investigated the usefulness of ALDH activity for characterizing HSC graft quality,particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time,and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly,in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells,this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism,whereas all other patients showed early complete donor chimerism. In conclusion,we suggest to measure ALDH activity as a surrogate marker for HSC activity,and to transport and store PBSC under controlled cooling conditions. View Publication

PTPN11 encodes the protein tyrosine phosphatase SHP-2,which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS),a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential,perturbed erythroid growth,and impaired normal differentiation in liquid cultures. In addition,leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments,which supports a primary role of hyperactive Ras in the pathogenesis of JMML. View Publication

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear,as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2,and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38,as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation,and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise,NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET,the positive regulator of IFN-gamma,and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC,and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively,our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC,and these effects are mediated via SMAD3. View Publication

Articular cartilage,which is mainly composed of collagen II,enables smooth skeletal movement. Degeneration of collagen II can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging. Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement. Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis." View Publication

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here,memory B cells are activated and amplified using Epstein-Barr virus infection,co-cultured with CHO-muCD40L cells,and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells,and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly,our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family,influenza A neutralizing antibodies,contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. View Publication

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis,and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here,we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus,a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus,this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome. View Publication