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

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections,and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2),which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs,HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However,HERVK is transcriptionally silenced by the host,with the exception of in certain pathological contexts such as germ-cell tumours,melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations,together with transactivation by OCT4 (also known as POU5F1),synergistically facilitate HERVK expression. Consequently,HERVK is transcribed during normal human embryogenesis,beginning with embryonic genome activation at the eight-cell stage,continuing through the emergence of epiblast cells in preimplantation blastocysts,and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably,we detected HERVK viral-like particles and Gag proteins in human blastocysts,indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product,the HERVK accessory protein Rec,in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection,suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover,Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy,indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. View Publication

Inhibition of metabolic features which distinguish cancer cells from their non-malignant counterparts is a promising approach to cancer treatment. Energy support for drug extrusion in multidrug resistance (MDR) is a potential target for metabolic inhibition. Two major sources of ATP-based metabolic energy are partial (glycolysis) and complete (mitochondrial oxidative phosphorylation) oxidation of metabolic fuels. In cancer cells,the balance between them tends to be shifted toward glycolysis; this shift is considered to be characteristic of the cancer metabolic phenotype. Numerous earlier studies,conducted with cells cultured in a monolayer (2-D model),suggested inhibition of glycolytic ATP production as an efficient tool to suppress MDR in cancer cells. Yet,more recent work challenged the appropriateness of the 2-D model for such studies and suggested that a more clinically relevant approach would utilize a more advanced cellular model such as a 3-D model. Here,we show that the transition from the 2-D model (cultured monolayer) to a 3-D model (cultured spheroids) introduces essential changes into the concept of energetic suppression of MDR. The 3-D cell organization leads to the formation of a discrete cell subpopulation (not formed in the 2-D model) with elevated MDR transport capacity. This subpopulation has a specific metabolic phenotype (mixed glycolytic/oxidative MDR support) different from that of cells cultured in the 2-D model. Finally,the shift to the oxidative phenotype becomes greater when the spheroids are grown under conditions of lactic acidosis that are typical for solid tumors. The potential clinical significance of these findings is discussed. View Publication

In spite of the recent improvements,the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer,a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here,we report on a component of a traditional Chinese medicine,evodiamine,which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase,it did not hold CSLC in a specific cell cycle phase but instead,selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly,evodiamine selectively activated p53 and p21 and decreased inactive Rb,the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. View Publication

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein,hiPSCs were differentiated into alpha-smooth muscle actin ($$-SMA) and calponin-positive VSMCs,which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted,which contained cells largely positive for $$-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover,when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts,they remained unruptured and patent with active vascular remodeling,and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs,and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. View Publication

It is well accepted that umbilical cord blood has been a source for hematopoietic stem cells. However,controversy exists as to whether cord blood can serve as a source of mesenchymal stem cells,which can differentiate into cells of different connective tissue lineages such as bone,cartilage,and fat,and little success has been reported in the literature about the isolation of such cells from cord blood. Here we report a novel method to obtain single cell-derived,clonally expanded mesenchymal stem cells that are of multilineage differentiation potential by negative immunoselection and limiting dilution. The immunophenotype of these clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Under appropriate induction conditions,these cells can differentiate into bone,cartilage,and fat. Surprisingly,these cells were also able to differentiate into neuroglial- and hepatocyte-like cells under appropriate induction conditions and,thus,these cells may be more than mesenchymal stem cells as evidenced by their ability to differentiate into cell types of all 3 germ layers. In conclusion,umbilical cord blood does contain mesenchymal stem cells and should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells to bone marrow. View Publication

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail,namely VCR (V,VPA,an inhibitor of HDACs; C,CHIR99021,an inhibitor of GSK-3 kinases and R,Repsox,an inhibitor of TGF-β pathways),under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs re- garding their proliferative and self-renewing abilities,gene expression profiles,and multipotency for different neu- roectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation,glycogen synthase kinase,and TGF-β pathways show similar efficacies for ciNPC induction. Moreover,ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemi- cal cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets,the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study,we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation,cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating,even after cell sheets were detached from culture dishes. Furthermore,extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. View Publication

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors,the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast,and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-$$B (NF-$$B) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-$$B was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover,both canonical and non-canonical NF-$$B signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact,canonical and non-canonical NF-$$B signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-$$B promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1$$ and interleukin-6. The results suggest the use of NF-$$B inhibitors for clinical therapy of certain breast cancers. View Publication

Asymmetry of cell fate is one fundamental property of stem cells,in which one daughter cell self-renews,whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms,such as plants,fungi,and mammals,has already been shown. However,before this current work,asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs),and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing,differentiating human and mouse ESCs. Moreover,we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3),indicating a molecular mechanism that regulates this phenomenon. Furthermore,our data support the hypothesis that retention of chromatids with the old" template DNA preserves the epigenetic memory of cell fate� View Publication

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts,and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer,retaining a pre-inactivation X chromosome state,and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF,naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells,they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression,pronounced tendency for X chromosome inactivation in most female human ES cells,increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells,from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells,and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively,our findings establish new avenues for regenerative medicine,patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. View Publication