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

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported,and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes,CD31 and CD144,in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells,freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes,marginal upregulation of CD31,and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however,no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters,which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage. View Publication

In the present study,we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women,13 non-pregnant women who had given birth to male offsprings,12 women who had never been pregnant,and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies� View Publication

The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study,constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML,expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression. View Publication

BACKGROUND: The purpose of this study was to determine whether murine mesenchymal stem cells (MSC) are able to home to the viable myocardium when injected intravenously and attenuate cardiac dysfunction and ventricular remodeling associated with myocardial infarction. METHODS AND RESULTS: Murine bone marrow cells were negatively selected for lineage markers and adherent MSC differentiated into adipocytes and osteocytes following treatment in culture. Two weeks after coronary occlusion that resulted in a permanent transmural infarct we observed a significant drop in LV systolic pressure,dP/dt(max),dP/dt(min),ESPVR and E(max) and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSC 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size,or end-diastolic volume. Injected MSC pre-labeled with fluorescent paramagnetic microspheres were observed scattered in noninfarcted regions of the myocardium. Flow cytometry of whole heart digests after intravenous injection of MSC labeled with either fluorescent microspheres or fluorescent PKH26 dye demonstrated that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart (20x) than those from uninfarcted controls. CONCLUSION: We conclude that intravenously injected MSC were able to home to viable myocardium and preserve systolic function by 2 weeks following ligation. The preserved contractility is likely an MSC-mediated paracrine response since infarct morphology was unchanged and labeled cells observed at two weeks exhibited the same characteristics as the injected MSC. These data underscore the importance of using MSC as a potential therapeutic intervention in preserving cardiac function following infarction. View Publication

PURPOSE To determine the safety,survival,and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN),group 2 (n = 59) received rCPCB-RPE1 implants,and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery),the eyes were examined histologically for cell survival,phagocytosis,and local toxicity. RESULTS Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry,suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS These results demonstrate the safety,survival,and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model. View Publication

Long noncoding RNAs (lncRNAs) exhibit diverse functions,including regulation of development. Here,we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition,we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans,and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm. View Publication

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. View Publication

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

BACKGROUND Autologous bone marrow mononuclear cell (ABMMNC) therapy has shown promise in patients with heart failure (HF). Cell function analysis may be important in interpreting trial results. METHODS In this prospective study,we evaluated the safety and efficacy of the transendocardial delivery of ABMMNCs in no-option patients with chronic HF. Efficacy was assessed by maximal myocardial oxygen consumption,single photon emission computed tomography,2-dimensional echocardiography,and quality-of-life assessment (Minnesota Living with Heart Failure and Short Form 36). We also characterized patients' bone marrow cells by flow cytometry,colony-forming unit,and proliferative assays. RESULTS Cell-treated (n = 20) and control patients (n = 10) were similar at baseline. The procedure was safe; adverse events were similar in both groups. Canadian Cardiovascular Society angina score improved significantly (P = .001) in cell-treated patients,but function was not affected. Quality-of-life scores improved significantly at 6 months (P = .009 Minnesota Living with Heart Failure and P = .002 physical component of Short Form 36) over baseline in cell-treated but not control patients. Single photon emission computed tomography data suggested a trend toward improved perfusion in cell-treated patients. The proportion of fixed defects significantly increased in control (P = .02) but not in treated patients (P = .16). Function of patients' bone marrow mononuclear cells was severely impaired. Stratifying cell results by age showed that younger patients (%60 years) had significantly more mesenchymal progenitor cells (colony-forming unit fibroblasts) than patients<60 years (20.16 ± 14.6 vs 10.92 ± 7.8,P = .04). Furthermore,cell-treated younger patients had significantly improved maximal myocardial oxygen consumption (15 ± 5.8,18.6 ± 2.7,and 17 ± 3.7 mL/kg per minute at baseline,3 months,and 6 months,respectively) compared with similarly aged control patients (14.3 ± 2.5,13.7 ± 3.7,and 14.6 ± 4.7 mL/kg per minute,P = .04). CONCLUSIONS ABMMNC therapy is safe and improves symptoms,quality of life,and possibly perfusion in patients with chronic HF. View Publication

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens,including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However,these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here,we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary,high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes,resulting in subsequent cholinergic neuron differentiation. Thus,our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells. View Publication