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

Due to widespread applications of human embryonic stem (hES) cells,it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation,and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture,we found out that hES cell recovery was significantly enhanced by around 30 % (P textless 0.05) by the new freezing solution. Moreover,at the first day of post-thaw culture,the presence of 10 microM ROCK inhibitor (Y-27632) and 1 microM pifithrin-mu together further significantly improved cell recovery by around 20% (P textless 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore,this protocol is a scalable cryopreservation method for handling large quantities of hES cells. View Publication

Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however,present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined,xeno-free,feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability,surface topography,surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content,have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture. View Publication

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this,time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis,including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique,in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work,and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality,fast chromosomal analysis of human ES and iPS cells. View Publication

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses,the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation,with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement,which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore,polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together,our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms. View Publication

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders,we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months,mice developed polycythemia,macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months,polycythemia regressed,abnormally shaped red blood cells and platelets were seen in circulation,and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia,thrombocytopenia,high neutrophilia,and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. View Publication

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors,genetic instability,and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells,transduced with these 2 ankyrin-1 promoter vectors,were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation,high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment),compared with negligible expression in myeloid and lymphoid lineages in blood,BM,spleen,and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus,modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer,stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases. View Publication

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast,very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC),although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL),Steel factor (SF),and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors,only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly,a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of textgreater 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However,this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL,SF,and IL-3. Also,for this response,the most potent single-acting factor tested was IL-3,not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols. View Publication

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity,coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity,which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function,and letrozole,an aromatase inhibitor,blocked androgen effects on telomerase activity. Conversely,flutamide,an androgen receptor antagonist,did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha),but not ER beta,inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use. View Publication

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture,purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population,compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse,suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. View Publication