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

Pre-clinical research on B and NK cell development relies on murine stromal cell-based systems with reduced physiological relevance and clinical applicability. A serum-free,fully humanized co-culture system utilizing human bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed to differentiate CB-CD34+ cells towards B and NK cell lineages. Differentiation dynamics were monitored via flow cytometry,with immunophenotypic analysis tracking progression from progenitors to mature cells. The system generated CD19+ IgM+ immature B cells and CD56+ CD16+ NK cells,recapitulating fetal stages of human lymphopoiesis. Serum-free media conditions ensured reproducibility and high overall yield of CD19+ B (35 ± 5.32%) and CD56+ NK (28.46 ± 7.01%) cell progenitors. Flow cytometry identified distinct population peaks,confirming temporal control over differentiation. This clinically relevant platform addresses the limitations of traditional models by providing a more physiologically accurate human microenvironment. The serum-free system supports applications in disease modeling,genotoxic compound screening,and mutational studies of hematopoiesis. By enabling scalable production of B and NK cells it aims to accelerate translational research for immunodeficiencies,cancer immunotherapy,and hematopoietic disorders. View Publication

Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However,the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells,and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential,which was elevated compared with control stromal cells,as indicated by the colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's],63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta2m-/- (NOD/SCID-beta2-/-) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results,and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta2m-/- RCs proliferated efficiently on Ihh stromal cells,compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells. View Publication

The main limitations of hematopoietic cord blood (CB) transplantation,viz,low cell dosage and delayed reconstitution,can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study,we use combinatorial cell culture technology (CombiCult{\textregistered}) to identify medium formulations that promote CD133+ CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133+ CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34+CD38lo/-CD45RA-CD90+CD49f+ population of hematopoietic stem cells and their differentiation into defined progeny. View Publication

Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades,embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article,we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system,which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases,it will be critical to establish a defined,reproducible and scalable culture system to meet the requirements of these clinical applications. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow,such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for textgreater or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described,but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues,we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor,interleukin (IL)-3,and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand,steel factor,IL-3,IL-6,granulocyte colony-stimulating factor,and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days,approximately 40% of which included textgreater or = 1 LTC-IC. In contrast,in similar cultures containing methylcellulose,input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications. View Publication

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication

To exploit the full potential of human pluripotent stem cells for regenerative medicine,developmental biology and drug discovery,defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed,but finding chemically defined,robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides,which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. View Publication

We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene,resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34(+) cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor,thrombopoietin,and Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34(+) cells,118-fold +/- 8-fold versus 117-fold +/- 13-fold; CFU-Cs,71-fold +/- 5-fold versus 67-fold +/- 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast,the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice,the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow. View Publication

BACKGROUND Aside from its importance in reproduction,estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However,the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). RESULTS E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-$$)-dependent pathway. During hematopoietic differentiation of hPSCs,ER-$$ is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-$$ positive cells. Interestingly,continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-$$ selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors,and subsequent erythropoiesis,whereas ER-$$ selective agonist did not. Furthermore,ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods,does E2 signaling potentiate hematopoietic development,suggesting universal function of E2 on hematopoiesis. CONCLUSIONS Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions. View Publication

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

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells,its physiological function(s) remain unknown,and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations,followed by competitive bone marrow reconstitution assays,shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent,the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore,PrP is a marker for HSCs and supports their self-renewal. View Publication