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

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment,young or old C57BL/6 bone marrow (BM) cells were injected,using a limiting-dilution,competitive transplantation method,into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age,the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly,the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However,the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage,indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes,erythrocytes,and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively,these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

BACKGROUND Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and also that insulators can improve FV vector safety. However,in a previous analysis of insulator effects on FV vector safety,strong viral promoters were used to elicit genotoxic events. In the present study,we developed and analyzed the efficacy and safety of a high-titer,clinically relevant FV vector driven by the housekeeping promoter elongation factor-1alpha$ and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). METHODS Human CD34+ cord blood cells were exposed to an enhanced green fluorescent protein expressing vector,FV-EGW-A1,at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. RESULTS FV-EGW-A1 resulted in high-marking,multilineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. CONCLUSIONS An FV vector with an elongation factor-1alpha$ promoter and an A1 insulator is a promising vector design for use in the clinic. View Publication

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication

Diffuse alveolar hemorrhage (DAH),although rare,is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans,although increasingly neutrophils,NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model,we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress,namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model,with accompanying reduction in IFN-driven genes and pathology. Lastly,coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH. View Publication

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs,focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations,gene expression,and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma,including severe type 2 inflammation,airway fibrosis,and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages,especially M2a and M2c. Furthermore,MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation,that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes,which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts,which were also suppressed by MSC treatment. In conclusion,intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management. View Publication

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study,we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation,the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry,immunoblotting,and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore,the cells were tested for functions associated with mature hepatocytes,including glycogen storage and indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate,numerous epithelial cells resembling hepatocytes were observed,and approximately 74% of the cells expressed the hepatic marker,albumin,after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers,and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development,and could provide a source of hepatocytes for transplantation therapy and drug screening. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication