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

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

Drug resistance and relapse are still major challenges in acute myeloid leukemia (AML) because of the inability to effectively eradicate leukemia stem cells (LSCs). Senescence induction combined with immune killing may offer promising strategies for LSC eradication. However,whether and how drug-resistant LSCs retain stemness via senescence and immune regulation remains unknown. The immunoproteasome subunit PSMB10 expression levels were analyzed by single-cell RNA-seq data,along with the bioinformatics analysis of publicly available AML datasets,and quantified using RT-qPCR and flow cytometry (FCM) analysis on clinical samples from AML patients. The cellular senescence was evaluated by the assays of cell proliferation,cell cycle,senescence-associated β-galactosidase activity,and senescence-associated secretory phenotype factors. In vitro T-cell killing assay was played to determine immune escape reprogramming of AML cells. FCM was conducted to estimate intracellular drug concentration and cellular apoptosis rates. Human AML xenografts and PSMB10 knockout syngeneic mouse bone marrow transplantation models were utilized to investigate the function of PSMB10. Various techniques were employed for mechanism studies,including Lentivirus transduction or siRNA transfection,western blotting,co-immunoprecipitation assays,luciferase reporter assays,polysome profiling assays,quantitative proteomics,etc. PSMB10 mRNA was significantly upregulated in the surviving nonsenescent LSCs,exhibiting a 13-fold increase compared to senescent LSCs following chemotherapy. The specific high expression of PSMB10 in post-chemotherapy nonsenescent LSCs predicts a poor AML prognosis. The genetic inactivation of PSMB10 resulted in increased senescence and cytotoxic T lymphocyte (CTL) killing,as well as increased intracellular drug concentrations and drug-induced cellular senescence in different types of human AML cells,which also impeded human and murine leukemia initiation and stemness maintenance in vivo with a 19-fold decrease in the frequency of human LSCs and a 7.6-fold decrease of drug-resistant mouse LSCs,while normal hematopoietic cells remained unaffected. Mechanistically,the downregulation of PSMB10 boosted SLC22A16-mediated drug endocytosis and further induced chemotherapy drug-mediated senescence through the RPL6/RPS6-MDM2-P21 pathway in AML cells. Additionally,downregulating PSMB10 also impeded MHC-I protein degradation-induced escape of CTL killing. PSMB10 is a key candidate molecular target for eradicating drug-resistant LSCs via senescence and immune regulation. The online version contains supplementary material available at 10.1186/s13046-025-03420-9. View Publication

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane,because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation,these methods are not standardized,require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA,or EDTA plus mechanical scraping with an electric toothbrush,or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding,and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2,γ1-γ3 chains,α1/α2 and α6 type IV collagen chains,fibronectin,nidogen-2,and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells,immortalized corneal epithelial cells,and induced pluripotent stem cells. This simple,fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients. View Publication

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here,we show that ectopic expression of miR-17,-20,-93 and -106,all AAAGUGC seed-containing miRNAs,increases proliferation,colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1),an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation,as a major target for these miRNAs in myeloid progenitors. In addition,we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further,SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment,but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion,replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. View Publication

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism,erythropoiesis,angiogenesis,and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene,which encodes the hypoxia-responsive alpha subunit of HIF-1,causes cardiac,vascular,and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos,as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines,such as vascular endothelial growth factor and Epo,but were ameliorated by Fe-SIH,a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis,Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin,Fpn1,Irp1,and frascati. We conclude that dysregulated expression of genes encoding Epo,EpoR,and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis. View Publication

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures,which show defective hematopoietic support as measured by production of total cells,granulocyte macrophage-colony-forming units (CFU-GMs),and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM,stromal mesenchymal precursors,fibroblast CFUs (CFU-Fs),and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover,IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1),Sca-1,CD49f,and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors,which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice. View Publication

We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte-colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33+ myeloid and CD14+ monocytic cells,whereas mobilized CD34+ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF-treated donors. c-suPAR was able to chemoattract CD34+ KG1 leukemia cells and CD34+ HSCs,as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR84-95). uPAR84-95 induced CD34+ KG1 and CD34+ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition,uPAR84-95 inhibited CD34+ KG1 and CD34+ HSC in vitro migration toward the stromal-derived factor 1 (SDF1),thus suggesting the heterologous desensitization of its receptor,CXCR4. Finally,uPAR84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34+ HSCs. Our findings demonstrate that G-CSF-induced upregulation of uPAR on circulating CD33+ and CD14+ cells is associated with increased uPAR shedding,which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization. View Publication

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase,SHIP-2,has been cloned [Pesesse,Deleu,De Smedt,Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239,697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines,spleen,thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover,SHIP-2 is detected in fibroblasts,heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells. View Publication

Individually,transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alter the growth and differentiation of normal and transformed osteoblast-like (OB) cells. Although recent evidence suggests interactions between TGF beta and 1,25(OH)2D3 may occur,little is known of the individual or combined effects of these hormones on the expression of the osteoblast phenotype at the cytochemical and biochemical levels in normal human OB (hOB) cells. Primary cultures of hOBs were treated with TGF beta (0.001-10 ng/ml) and 1,25(OH)2D3 (0.1 pM-100 nM) either alone or in combination. TGF beta and 1,25(OH)2D3 stimulated spindle-shaped cells to become stellate in appearance and increased the number of cytoplasmic processes. TGF beta increased 3H-thymidine incorporation and 1,25(OH)2D3 reduced this effect. Conversely,procollagen type-I synthesis and secretion were increased in a dose-dependent manner in the presence of TGF beta but were not significantly affected in the presence of 1,25(OH)2D3. TGF beta and 1,25(OH)2D3 each marginally increased alkaline phosphatase (ALP) activity,but the combination synergistically increased ALP activity in a dose- and time-dependent manner at the cytochemical and biochemical level (three to tenfold over vehicle controls; n = 12). In contrast,TGF beta reduced 1,25(OH)2D3-stimulated osteocalcin secretion. These data suggest that TGF beta stimulates hOB cells to actively produce collagen matrix and proliferate. The combination of TGF beta and 1,25(OH)2D3,however,produces a synergistic increase in ALP activity and maintenance of collagen synthesis. 1,25(OH)2D3 stimulation may induce cells to advance to an endstage where cell proliferation is reduced and osteocalcin expression is promoted. Interactions between TGF beta and 1,25(OH)2D3 may represent important steps in the regulation of osteoblast differentiation and matrix production. View Publication