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

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here,we further dissected the ALDH(hi)-Lin- population by selection for CD133,a surface molecule expressed on progenitors from hematopoietic,endothelial,and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+,but also included CD34-CD38-CD133+ cells,a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro,whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells,suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial,secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors,detected as long-term culture-initiating cells (LTC-IC),could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC,CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold,respectively,above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition,stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms. View Publication

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here,we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state,followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. View Publication

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction,and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC,ending with development of procedures for banking. First,we aimed to optimize cryopreservation of established DPSC cultures,with regards to optimizing the cryoprotective agent (CPA),the CPA concentration,the concentration of cells frozen,and storage temperatures. Secondly,we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly,we evaluated the growth,surface markers and differentiation properties of DPSC obtained from intact teeth and undigested,whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen,at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues,with digestion and culture performed post-thaw. A recovery of cells from textgreater85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free,defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions. View Publication

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However,an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study,we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1,side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore,knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice,supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion,but not for CSC maintenance. Taken together,our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus,targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer. View Publication

For antibody-based therapy of cancer,monoclonal antibodies (mAbs) of human origin are superior to mouse,mouse/human chimeric or humanized mAbs,because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study,human mAbs were prepared against a pancarcinoma antigen,MK-1 (Ep-CAM),using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice,immunized with recombinant MK-1,were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed,two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants,their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity,respectively,against MK-1-expressing cells in vitro,suggesting that these fully human mAbs produced against MK-1 and their V-region genes,which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer. View Publication

ETS2 and ERG are transcription factors,encoded on human chromosome 21 (Hsa21),that have been implicated in human cancer. People with Down syndrome (DS),who are trisomic for Hsa21,are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1,which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis,we expressed ETS2,ERG,and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2,ERG,and FLI-1 facilitated the expansion of megakaryocytes from wild-type,Gata1-knockdown,and Gata1s knockin progenitors,but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors,their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin,but not wild-type,fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway,commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis. View Publication

Although prolonged transgene expression in progenitor cells might be desirable for modified cell therapy,the viral promoter-based expression vector tends to promote transgene expression only for a limited period. Here,we examined the ability of cellular promoters from elongation factor-1alpha (EF-1alpha) and ubiquitin C to drive gene expression in hematopoietic TF-1 and mesenchymal progenitor cells. We compared the expression levels and duration of a model gene,interleukin-2,generated by the cellular promoters to those by the cytomegalovirus (CMV) promoter. The EF-1alpha and ubiquitin C promoters drove prolonged gene expression in hematopoietic TF-1 and mesenchymal progenitor cells,whereas the CMV promoter did not. At day 7 after transfection in TF-1 cells,the mRNA expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 118- and 56-fold higher,respectively,than those driven by the CMV promoter. Similarly,in mesenchymal progenitor cells,the expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 98- and 20-fold higher,respectively,than that driven by the CMV promoter-encoding plasmid. Moreover,the ubiquitin C promoter directed higher levels of green fluorescence protein expression in mesenchymal progenitor cells than did the CMV promoter. These results indicate that the use of cellular promoters such as those for EF-1alpha and ubiquitin C might direct prolonged gene expression in hematopoietic and mesenchymal progenitor cells. View Publication

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA),we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-$\gamma$ for 72??h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g.,CD48,CD74,and CD86) in DR(-) cells,which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens. View Publication