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

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

Label-retaining cancer cells (LRCCs) represent a novel population of stem-like cancer cells exhibiting slow cycling,chemoresistance and tumor-initiating capacities; however,their properties remain unclear,and approaches to eradicate LRCCs remain elusive. Here,we report that colon cancer cells with high fluorescent intensity,referred to as LRCCs,have the greatest cancer stem cell (CSC)-like capacities and that they preferentially express CSC markers and stemness-related genes. Moreover,we found that Lgr5,which has been reported to be a marker of rapid cycling CSCs,is almost negatively expressed in LRCCs but that its expression is gradually increased in the differentiation process of LRCCs. Interestingly,we found that LRCCs are especially sensitive to the pro-apoptotic effect of IFN-γ treatment both in vitro and in vivo because LRCCs possess higher IFN-γR levels compared with non-LRCCs,which results in the upregulation of the apoptosis pathway after IFN-γ treatment. Furthermore,we found that IFN-γ shows synergistic effects with the conventional anticancer drug Oxaliplatin to eliminate both LRCCs and non-LRCCs. In conclusion,this is the first study to suggest that LRCCs,as a distinct tumor-initiating population,can be selectively eradicated by IFN-γ,which may provide a novel therapeutic strategy for colon cancer treatment. View Publication

The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation,but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation,induces cell-cycle arrest in G(1) phase,and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors,and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB,facilitating megakaryocyte differentiation,and of CDK4 and CDK6,to inhibit the G(1)/S transition. However,these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However,in p53-null K562 cells,phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript. 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

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors,the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors,however,may be needed to guide cell growth and differentiation. To provide access to such new materials,we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait,approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested,and seven cell-binding peptides were identified. Of these,six sequences possess EC cell-binding ability. Specifically,when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold,they mediate cell adhesion. The corresponding soluble peptides block this adhesion,indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR),they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells. 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

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian,Botryllus schlosseri,vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues,gametes,or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent,self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates,but not to both,is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele,these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance. View Publication

It is well known that hepatic stellate cells (HSC) develop into cells,which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that beta-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119,an inhibitor of the glycogen synthase kinase 3beta,led to reduced beta-catenin phosphorylation,induced nuclear translocation of beta-catenin,elevated glutamine synthetase production,impeded synthesis of alpha-smooth muscle actin and Wnt5a,but promoted the expression of glial fibrillary acidic protein,Wnt10b,and paired-like homeodomain transcription factor 2c. In addition,canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that beta-catenin-dependent Wnt signaling maintains the quiescent state of HSC and,similar to stem and progenitor cells,influences their developmental fate. View Publication

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs,or treatment with recombinant BMP4,with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin,a BMP antagonist,demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore,BECs may provide a feedback mechanism to control the proliferation of NSPCs. View Publication