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

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

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

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

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

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

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

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology,gene therapy,and clinical transplantation. Here,we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor,thrombopoietin,Flt-3 ligand,interleukin (IL)-3,and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition,we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice,which showed myeloid,B,T,and natural killer cells as well as CD133(+)CD34(+) cells,and hematopoietic reconstitution in the secondary recipients. Interestingly,the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation. View Publication

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382,p53p),linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction,induced rapid apoptosis in human premalignant and malignant cell lines. Here,we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas,which express sufficient quantities of endogenous mutant or WT p53,may be restored or activated,respectively,by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system,which circumvents the blood-brain barrier. View Publication

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However,the current conventional method leads to poor recovery of hPSCs after thawing. Here,we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing,we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival,whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover,the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication