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

Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998),human embryonic stem cells (hESCs) have been derived from blastocysts,and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs,fibroblast feeder layers are often used at some phase in the culturing protocol. The feeders - often mouse embryonic fibroblasts (mEFs) - provide a substrate that increases plating efficiency,helps maintain pluripotency,and facilitates survival and growth of the stem cells. Various protocols for culturing embryonic stem cells from both species are available with newer trends moving toward feeder-free and serum-free culture. The purpose of this chapter is to provide basic protocol information on the isolation of mouse embryonic fibroblasts and establishment of feeder layers,the culture of mESCs on both mEFs and on gelatin in serum-containing medium,and the culture of hESCs in defined media on both mEFs (hESC culture medium) and Matrigel (mTeSR). These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose. View Publication

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

BACKGROUND The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application. METHODS Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium,Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated. RESULTS We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF,respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF,respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium,for two cryopreservation systems (Ptextless0.05). CONCLUSION We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated,maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies. View Publication

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br),and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations),to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD,n=30) nucleated cells and was enriched in cells expressing CD34,CD117,CD105,CD127,CD133 and CD166,and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells,and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes,osteoblasts and chondrocytes. DISCUSSION: Hematopoietic,endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. 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

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication

PURPOSE: Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells,but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. METHODS: The seven osteosarcoma cell lines Cal72,SJSA-1,Saos-2,SK-ES-1,U2OS,143.98.2,and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). RESULTS: Although proliferation often was relatively low in serum-free media (X-vivo 10,X-vivo 15,X-vivo 20,Stem Span SFEM),some cell lines (Cal72,KHOS-32IH,Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However,all cell lines proliferated well in Stem Span with FCS,and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS),and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72,SJSA-1),and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. CONCLUSIONS: Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines,but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation,cytokine release); and (iii) whether coculture experiments are included. View Publication

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling,we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor,p21Cip1/Waf1 (p21),have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling,we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control,manipulated cells. Further,we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore,lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. View Publication

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected,we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs,where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection,as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability,expansion,and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro,it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether,these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression. View Publication

In previous reports,we have demonstrated that only direct cell-cell contact with stromal cells,such as the murine stromal cell line AFT024,was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment,we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose,we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours,human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g.,fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC,thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism. View Publication