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

Femtosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy offers several advantages over spontaneous Raman spectroscopy due to the inherently high sensitivity and low average power deposition in the sample. Femtosecond CARS can be implemented in a collinear pump/probe beam configuration for microspectroscopy applications and has emerged as a powerful technique for chemical imaging of biological specimens. However,one serious limitation of this approach is the presence of a high nonresonant background component that often obscures the resonant signals of interest. We report here an innovative pulse-shaping method based on Lorentzian amplitude and phase spectral modulation of a broadband femtosecond probe pulse that yields spectra with both high spectral resolution and no nonresonant background. No further mathematical analysis is needed to extract Raman spectra. The utility of the proposed method for CARS microscopy is demonstrated using a mixture of polystyrene and latex beads,as well as dry-fixed embryonic stem cells. View Publication

BACKGROUND Congenital human cytomegalovirus (HCMV) infection,a leading cause of birth defects,is most often manifested as neurological disorders. The pathogenesis of HCMV-induced neurological disorders is,however,largely unresolved,primarily because of limited availability of model systems to analyze the effects of HCMV infection on neural cells. METHODS An induced pluripotent stem cell (iPSC) line was established from the human fibroblast line MRC5 by introducing the Yamanaka's four factors and then induced to differentiate into neural stem/progenitor cells (NSPCs) by dual inhibition of the SMAD signaling pathway using Noggin and SB-431542. RESULTS iPSC-derived NSPCs (NSPC/iPSCs) were susceptible to HCMV infection and allowed the expression of both early and late viral gene products. HCMV-infected NSPC/iPSCs underwent apoptosis with the activation of caspase-3 and -9 as well as positive staining by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Cytochrome c release from mitochondria to cytosol was observed in these cells,indicating the involvement of mitochondrial dysfunction in their apoptosis. In addition,phosphorylation of proteins involved in the unfolded protein response (UPR),such as PKR-like eukaryotic initiation factor 2a kinase (PERK),c-Jun NH2-terminal kinase (JNK),inositol-requiring enzyme 1 (IRE1),and the alpha subunit of eukaryotic initiation factor 2 (eIF2$$) was observed in HCMV-infected NSPC/iPSCs. These results,coupled with the finding of increased expression of mRNA encoding the C/EBP-homologous protein (CHOP) and the detection of a spliced form of X-box binding protein 1 (XBP1) mRNA,suggest that endoplasmic reticulum (ER) stress is also involved in HCMV-induced apoptosis of these cells. CONCLUSIONS iPSC-derived NSPCs are thought to be a useful model to study HCMV neuropathogenesis and to analyze the mechanisms of HCMV-induced apoptosis in neural cells. View Publication

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study,we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB),although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction,the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition,the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies,whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore,an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population,and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB,selection by AC133 expression is better than selection by CD34 expression. View Publication

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis,we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8),which induces a myeloproliferation in vivo. Among the targeted genes,we identified Mef2c,encoding a MCM1-agamous-deficiens-serum response factor transcription factor,and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly,several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions,prompting an investigation of the causal interplay. Using a conditional mouse strain,we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however,its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors,providing a mechanistic link to increased homing and motility. Thus,MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype. View Publication

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE,which encodes neutrophil elastase (NE). However,a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end,we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs),and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest,and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly,high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBP$$-dependent emergency granulopoiesis. In contrast,sivelestat,an NE-specific small-molecule inhibitor,corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA,but not CEBPB; and promoting promyelocyte survival and differentiation. Together,these data suggest that SCN disease pathogenesis includes NE mislocalization,which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization. View Publication

BACKGROUND: Vascular trauma induced by surgical revascularization stimulates mobilization of hematopoietic and nonhematopoietic progenitor cells. However,it is not clear whether mobilized progenitors are functionally active and participate in peripheral homing. We have found no clinical studies available regarding the reaction of bone marrow to surgical revascularization. METHODS: This was an observational prospective study of 76 patients undergoing elective coronary artery bypass graft surgery. Bone marrow aspirates and blood samples were collected at baseline,at the end of surgery,and 24 hours postoperatively (blood samples only). The CD34+,CD34+CD133+,and CD34+CXCR4+ progenitor cell counts,CXCR4+ mononuclear cell counts,and CXCR4 expression on CD34+ cells were measured by flow cytometry. Progenitor cell functions were studied in vitro by clonogenic and migration assays. RESULTS: In response to coronary revascularization there was mobilization of CD34+ progenitors,having increased migratory and clonogenic function. The CD34+CXCR4+ subsets and CXCR4 expression on CD34+ cells in peripheral blood increased significantly 24 hours postoperatively. The CXCR4 expression on mobilized progenitors at the end of surgery was independently related to baseline CXCR4 expression on bone marrow resident CD34+ cells and duration of cardiopulmonary bypass in a multivariate model. At the end of surgery there was a significant fall in the expression of CXCR4 on CD34+ bone marrow cells,suggesting egress into peripheral circulation of the most active CXCR4-expressing progenitors. CONCLUSIONS: Coronary artery bypass graft surgery is associated with bone marrow release of functionally active progenitor cells. Further studies are needed to verify whether mobilized progenitors participate in regeneration of injured tissues. View Publication

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here,we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages,respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations,suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs,which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells. View Publication

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield. View Publication

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity,in contrast to CD133(+)G(1) cells. Here,we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%,respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures,the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (Ptextless0.001) throughout the culture period. Furthermore,a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols. View Publication

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However,it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here,we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions,revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions,pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors,or from Oct4 alone,resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover,the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research. View Publication

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties,we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR,a fluorescent substrate for ALDH,and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells,but,we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly,ALDH activity and Aldh1a1 expression levels were very low in mouse,rat,rabbit and non-human primate myoblasts. Using different approaches,from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde,an inhibitor of class I ALDH,to cell fractionation by flow cytometry using the ALDEFLUOR assay,we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity,as a purification strategy,could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. View Publication