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

The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10,X-vivo 15,[Bio-Whitacker,Walkersville,MD] and StemSpan [Stem Cell Technologies,Vancouver,BC,Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used,and clonogenic cells were maintained after in vitro culture in all media. In contrast,constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines,but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines. View Publication

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy,we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters,the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters,suggesting a relative resistance to silencing. Furthermore,an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector,largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile. View Publication

Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells,the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here,we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ,expression of p16(INK4a) is upregulated in progenitor and stem cells,inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence. View Publication

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such,hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However,for their full potential to be realized,both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however,they suffer from some major limitations including lack of definition,xenobiotic nature,batch-to-batch variation,and labor-intensive production. Therefore,hESC culture definition is essential if hESC lines,and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state,for over 30 passages using MT and SP. Additionally,we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture,contributing to the standardization of hESC in vitro models and ultimately their application. View Publication

SummaryHuman pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment.For complete details on the use and execution of this protocol,please refer to Wells et al.1 Graphical abstract Highlights•Brief pulse of Ngn2 induces neural progenitor cells from human stem cells•Guidance on expanding,freezing,and thawing SNaP cells for future use•Immunostaining-based assays assess cell identity and differentiation potential Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment. View Publication

Human pluripotent stem cells (hPSCs) and their differentiated derivatives represent valuable tools for studying development,modeling diseases,and advancing cell therapy. Recent improvements in genome engineering allow for precise modifications of hPSCs,further enhancing their utility in basic and translational research. Here we describe a Recombinase-Mediated Cassette Exchange (RMCE) platform in hPSCs that allows for the highly efficient,rapid,and specific integration of transgenes. The RCME-mediated DNA integration process is nearly 100% efficient,without negatively affecting the pluripotency or karyotypic stability of hPSCs. Taking advantage of this convenient system,we first established a dual inducible expression system based on the Tet-On and Cumate-On systems,allowing for the inducible expression of two transgenes independently. Secondly,we incorporated a Tet-on inducible system,driving the expression of three genes simultaneously. However,two genes also contain independent degron sequences,allowing for precise control over the expression of each gene individually. We demonstrated the utility of these systems in hPSCs,as well as their functionality after differentiation into cells that were representative of the three germ layers. Lastly,we used the triple inducible system to investigate the lineage commitment induced by the pancreatic transcription factors NKX6.1 and PDX1. We found that controlled dual expression,but not individual expression,biases hPSC embryoid body differentiation towards the pancreatic lineage by inducing the expression of the NeuroD program. In sum,we describe a novel genetic engineering platform that allows for the efficient and fast integration of any desired transgene(s) in hPSCs using RMCE. We anticipate that the ability to modulate the expression of three transgenes simultaneously will further accelerate discoveries using stem cell technology. View Publication

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However,there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein,we provide a detailed protocol for the production,enrichment,and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol,researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines,researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks,and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis,as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease,$\beta$-thalassemia,and primary immunodeficiencies. View Publication

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However,these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation,thereby limiting their range of applications. In this protocol,we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA). View Publication

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically,it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g.,peptides,proteins,or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion. View Publication

Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues,such as stem cells and cancer tumors. However,a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability,confounds studies with therapeutic outcomes for cultured cells,and represents a major cost associated with cell culture. Here we report a commercially available serum-free,albumin-free,and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages,fibroblasts,as well as specific cancer cell lines; without the use of contaminants such serum,albumin,and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture. View Publication

AIMS The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro,and test the effect of in vivo environments on the further maturation of hESCs derived cells. MAIN METHODS Embryoid bodies (EBs) formed from hESCs,with serum-free KSR-based medium and mesodermal specification related factors,CHIR,and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB,TGF β3,BMP4 in sequence for 24days. The morphology,FACS,histological staining as well as the expression of chondrogenic specific genes were detected in each stage,and further in vivo experiments,cell injections and tissue transplantations,further verified the formation of chondrocytes. KEY FINDINGS We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule,their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. SIGNIFICANCE The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes,and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. View Publication

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk,a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability,clonogenic hematopoietic potential,and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells,together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel,the supportive cells self-assemble into a hypoxic stromal network,stimulating CD34+ CD38+ cell formation,while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks,in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly,the primary in vitro niche model supports HSPCs with no cytokine addition. Overall,the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches,in the context of normal hematopoiesis or blood-related diseases. View Publication