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

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that,unlike steady-state erythropoiesis,erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM),severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments,donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context,stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system,EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion,and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x),in contrast,was not significantly induced via WT-EpoR,EpoR-HM,or EpoR-H alleles. In Kit+ CD71+ erythroblasts,EpoR-PY343 signals furthermore enhanced SCF growth effects,and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts,oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis,therefore,requires stage-specific EpoR-PY343-Stat5 signals,some of which selectively bolster SCF and oncostatin-M action. View Publication

Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence,or minimize expression,of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming,we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression. View Publication

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders,highlighted by their successful therapeutic use in inherent immunodeficiencies. However,biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here,we report an RNA-based episomal vector system,amenable for long-term transgene expression in stem cells. Specifically,we used a unique intranuclear RNA virus,Borna disease virus (BDV),as the gene transfer vehicle,capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology,cell surface CD105 expression,or the adipogenicity of MSCs. Similarly,replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs),while maintaining the ability to differentiate into three embryonic germ layers. Thus,the BDV-based vectors offer a genomic modification-free,episomal RNA delivery system for sustained stem cell transduction.Gene Therapy accepted article preview online,03 December 2015. doi:10.1038/gt.2015.108. View Publication

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However,the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here,we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further,we investigated the transcriptional changes in mRNA and miRNA levels,using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs. View Publication

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical,physiological,cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular,it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species,including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust,allowing for morphological visualization,activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species,thus opening the possibility to study GABAergic function in virtually any vertebrate species. View Publication

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme,thereby correcting the biochemical defect and halting disease progression. Here,we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels,maintain long-term repopulation and multi-lineage differentiation potential,and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders. View Publication

In this unit,we describe a simple coculture-free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow-derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured,the difficulty in introducing specific genetic changes into these cells,and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cells from embryonic stem cells addresses these limitations. ES-derived mast cells can be generated in numbers sufficient for studies of the pathways involved in mast cell effector functions. These ES cell-derived mast cells respond to antigens and other stimuli by releasing histamine,cytokines,lipids,and other bioactive mediators. The derivation of human mast cells from ES cells carrying mutations introduced by homologous recombination should provide a novel means of testing the function of genes in both the development and the effector functions of mast cells. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice,with a corresponding retention of progenitors in blood. Remarkably,the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells,studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin,studied in parallel. Furthermore,the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies,in contrast to antibodies against alpha4 integrin,did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells. View Publication

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages,characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages,we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly,these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells,GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly,F4/80 expression in macrophages was suppressed in the homozygous mutant,although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells,MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages,whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation,renal tubule survival,and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages. View Publication