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

BACKGROUND Pluripotency,the property of a cell to differentiate into all cellular types of a given organism,is central to the development of stem cell-based therapies and regenerative medicine. Stem cell pluripotency is the result of the orchestrated activation of a complex transcriptional network characterized by the expression of a set of transcription factors including the master regulators of pluripotency Nanog and Oct4. Recently,it has been shown that pluripotency can be induced in somatic cells by viral-mediated expression of the transcription factors Oct3/4,Sox2,Klf4,and c-Myc. RESULTS Here we show that 5-Aminoimidazole-4-carboxamide-1-b-riboside (AICAR) is able to activate the molecular circuitry of pluripotency in mouse embryonic stem cells (mESC) and maintain Nanog and Oct4 expression in mESC exposed to the differentiating agent retinoic acid. We also show that AICAR is able to induce Klf4,Klf2 and Myc expression in both mESC and murine fibroblasts. CONCLUSION AICAR is able to activate the molecular circuitry of pluripotency in mESC and to induce the expression of several key regulators of pluripotency in somatic cells. AICAR is therefore a useful pharmacological entity for studying small molecule mediated induction of pluripotency. View Publication

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome,formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment,we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method,we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly,we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+),whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations,we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition,by generating an anti-IL1RAP antibody,we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML. View Publication

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar theratype"; however a standardized approach confirming efficacy in these cohorts has not been reported. Here we demonstrate that patients bearing the rare mutation: c.3700 A>G causing protein misprocessing and altered channel function-similar to ΔF508-CFTR are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor respectively this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound effective in increasing the levels of immature CFTR protein augmented the Orkambi® response. Importantly this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue will facilitate therapy development for patients with rare CF mutations. View Publication

Objectives gamma$delta$ T cells,a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens,are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation,we compared how exposure to an autoantigenic lipid,cardiolipin (CL),during co-stimulation with an amino-bisphosphonate (zoledronate,zol),affects the activation and cytokine production of SSc and healthy control (HC) gamma$delta$ T cells. Methods Expression of CD25 on Vgamma$9+,Vdelta$1+,and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs),their binding of CD1d tetramers,and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNgamma$ and IL-4 was assessed after overnight culture. Results Percentages of CD25+ among CD3+ and Vdelta$1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC,CL and zol,respectively,suppressed {\%}CD25+ Vgamma$9+ and Vdelta$1+ T cells but,when combined,CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly,Vdelta$1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers,and a CD1d-blocking mAb decreased CL-induced enhancement of {\%}SSc CD25+ Vdelta$1+ T cells in the presence of zol. {\%}IFNgamma$+ cells among Vgamma$9+ T cells of SSc was lower than HC cultured in medium,CL,zol,or CL + zol,whereas {\%}IFNgamma$+ Vdelta$1+ T cells was lower only in the presence of CL or CL + zol. {\%}IL-4+ T cells were similar in SSc and HC in all conditions,with the exception of being increased in SSc Vgamma$9+ T cells in the presence of CL. Conclusion Abnormal functional responses of gamma$delta$ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression,characteristics of this disease. View Publication

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures,derived from isolated single cells,can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells,or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo,including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development. View Publication

OBJECTIVE: To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell (CML-LSC) and its mechanism. METHODS: CD34(+)CD38(-)CD123(+) leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia (CML) patients BM cells and CD34(+)CD38(-) hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySep(TM) magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM),and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay,respectively. RESULTS: (1) After incubated with IM7,the LSC and HSC CD44 expression rates were (86.60 ± 2.10)% vs. (25.40 ± 1.70)% (P textless 0.05),respectively. (2) The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7,the OD value (A(570)) changing from pre-incubation of (0.62 ± 0.11) to post-incubation of (0.34 ± 0.07),while there was little change of A(570) in the HSC group. (3) The migration ability of the LSC group was inhibited evidently after incubated with IM7,the inhibition rate being 46% ∼ 63%,while little change of that in HSC group was detected. (4) The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7,while little change was found in that of HSC group. CONCLUSION: The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro,which might provide a theoretical evidence for targeting therapy. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

BACKGROUND Mesenchymal stem cells (MSCs) have immense self-renewal capability. They can be differentiated into many cell types and therefore hold great potential in the field of regenerative medicine. MSCs can be converted into beating cardiomyocytes by treating them with DNA-demethylating agents. Some of these compounds are nucleoside analogs that are widely used for studying the role of DNA methylation in biological processes as well as for the clinical treatment of leukemia and other carcinomas. AIMS To achieve a better therapeutic option for cardiovascular regeneration,this study was carried out using MSCs treated with two synthetic compounds,zebularine and 5-azacytidine. It can be expected that treated MSCs prior to transplantation may increase the likelihood of successful regeneration of damaged myocardium. METHODS The optimized concentrations of these compounds were added separately into the culture medium and the treated cells were analyzed for the expression of cardiac-specific genes by RT-PCR and cardiac-specific proteins by immunocytochemistry and flow cytometry. Treated MSCs were cocultured with cardiomyocytes to see the fusion capability of these cells. RESULTS mRNA and protein expressions of GATA4,Nkx2.5,and cardiac troponin T were observed in the treated MSCs. Coculture studies of MSCs and cardiomyocytes have shown improved fusion with zebularine-treated MSCs as compared to untreated and 5-azacytidine-treated MSCs. CONCLUSION The study is expected to put forth another valuable aspect of certain compounds,that is,induction of transdifferentiation of MSCs into cardiomyocytes. This would serve as a tool for modified cellular therapy and may increase the probability of better myocardial regeneration. View Publication

Background: The complex aetiology of type 1 diabetes (T1D),characterised by a detrimental cross-talk between the immune system and insulin-producing beta cells,has hindered the development of effective disease-modifying therapies. The discovery that the pharmacological activation of LRH-1/NR5A2 can reverse hyperglycaemia in mouse models of T1D by attenuating the autoimmune attack coupled to beta cell survival/regeneration prompted us to investigate whether immune tolerisation could be translated to individuals with T1D by LRH-1/NR5A2 activation and improve islet survival. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from individuals with and without T1D and derived into various immune cells,including macrophages and dendritic cells. Cell subpopulations were then treated or not with BL001,a pharmacological agonist of LRH-1/NR5A2,and processed for: (1) Cell surface marker profiling,(2) cytokine secretome profiling,(3) autologous T-cell proliferation,(4) RNAseq and (5) proteomic analysis. BL001-target gene expression levels were confirmed by quantitative PCR. Mitochondrial function was evaluated through the measurement of oxygen consumption rate using a Seahorse XF analyser. Co-cultures of PBMCs and iPSCs-derived islet organoids were performed to assess the impact of BL001 on beta cell viability. Results: LRH-1/NR5A2 activation induced a genetic and immunometabolic reprogramming of T1D immune cells,marked by reduced pro-inflammatory markers and cytokine secretion,along with enhanced mitohormesis in pro-inflammatory M1 macrophages and mitochondrial turnover in mature dendritic cells. These changes induced a shift from a pro-inflammatory to an anti-inflammatory/tolerogenic state,resulting in the inhibition of CD4+ and CD8+ T-cell proliferation. BL001 treatment also increased CD4+/CD25+/FoxP3+ regulatory T-cells and Th2 cells within PBMCs while decreasing CD8+ T-cell proliferation. Additionally,BL001 alleviated PBMC-induced apoptosis and maintained insulin expression in human iPSC-derived islet organoids. Conclusion: These findings demonstrate the potential of LRH-1/NR5A2 activation to modulate immune responses and support beta cell viability in T1D,suggesting a new therapeutic approach. Key points: LRH-1/NR5A2 activation in inflammatory cells of individuals with type 1 diabetes (T1D) reduces pro-inflammatory cell surface markers and cytokine release. LRH-1/NR5A2 promotes a mitohormesis-induced immuno-resistant phenotype to pro-inflammatory macrophages. Mature dendritic cells acquire a tolerogenic phenotype via LRH-1/NR5A2-stimulated mitochondria turnover. LRH-1/NR5A2 agonistic activation expands a CD4+/CD25+/FoxP3+ T-cell subpopulation. Pharmacological activation of LRH-1/NR5A2 improves the survival iPSC-islets-like organoids co-cultured with PBMCs from individuals with T1D. View Publication

Introduction: The maintenance of intestinal homeostasis depends on a complex interaction between the immune system,intestinal epithelial barrier,and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover,persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7),which recognizes single-stranded RNA viruses,and ATG16L1,which facilitates the delivery of viral nucleic acids to the autolysosome endosome,are required for anti-viral immune responses. Results and discussion: However,the role of the enteric virome in IBD is still poorly understood. Here,we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state,Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice,reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore,Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index,histoscore,and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. Conclusion: In conclusion,the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants. View Publication

Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet,since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling,it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Serial modifications to an existing iMGL protocol were made,including but not limited to changes in growth factor combination to drive microglial differentiation,until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines,the quality of the new iMGL protocol was validated through cell yield assessment,measurement of microglia marker expression,transcriptomic comparison to primary microglia,and evaluation of inflammatory and phagocytic activities. Similarly,molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol,decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally,ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression,a heightened capacity to internalize myelin,as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency,as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL,and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. We optimized a pre-existing iMGL protocol,generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol,we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant,with preliminary characterization pointing toward functional alterations in migratory,phagocytic and inflammatory activities. The online version contains supplementary material available at 10.1186/s13024-024-00723-x. View Publication

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4 + T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling,resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML,serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro . Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore,promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML. View Publication