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

Simvastatin,a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor,was used in cardiovascular diseases and could decrease low-density lipoprotein cholesterol,and may have a repurposed role in cancer therapy. However,the effects of simvastatin on endometrial cancer remain controversial. We aimed to elucidate the role and mechanisms of simvastatin in regulating previously identified endometrial cancer-associated mesenchymal stem cells (EmCaMSCs)-mediated immunosuppressive effects and anti-tumor progression. Coculture of EmCaMSCs and peripheral blood mononuclear cells (PBMC) was used to assay the population of CD8 + T cells,natural killer (NK) cells,and cytotoxicity of NK cells. The mechanisms were elucidated by applying recombinant proteins and inhibitors of candidate proteins,transforming growth factor-beta 2 (TGF-β2). Finally,the humanized mouse model was generated to study the effects of simvastatin-mediated immunotherapy in treating endometrial cancer. The protein expressions of TGF-β2,CD56,CD8,and PD-L1 in xenograft tumors were analyzed by Western blot or immunohistochemistry assay. In this study,simvastatin inhibited the proliferation of endometrial cancer cells (HEC-1 A and RL95-2) and EmCaMSCs,and the half-maximal inhibitory concentration (IC50) values of EmCaMSCs were much higher. Simvastatin rescued the proliferation and the population of CD8 + T cells and natural killer (NK) cells from PBMC coculturing with EmCaMSC. Simvastatin treatment reduced the expression of TGF-β2 in EmCaMSCs at both the gene and protein levels. TGF-β2 activated the downstream SMAD2/3 signaling,and their inhibition by simvastatin could enhance the cytotoxicity of NK cells against endometrial cancer cells in vitro. Additionally,a combination of simvastatin and NK cell therapy inhibited xenograft growth,potentially by reducing TGF-β2 expression. In conclusion,simvastatin could rescue the population of CD8 + T cells and NK cells from PBMC cocultured with EmCaMSCs. Furthermore,simvastatin could enhance the cytotoxicity of NK cells in vitro and inhibit tumor growth in vivo in a humanized mouse model. These results suggested that simvastatin may be considered as a repurposed and combination drug for treating endometrial cancer. The online version contains supplementary material available at 10.1038/s41598-025-08686-9. View Publication

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development,molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells,indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame,suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast,in alphabeta cells,the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were,however,depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell,suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34,and plateaued at the early double positive stage. Surprisingly,however,some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway,an instructional role for the TCR in the alphabeta/gammadelta lineage choice,and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci,the timing of beta selection,and maintenance of gammadelta potential through the early double positive stage of development. View Publication

The ST6Gal-I sialyltransferase adds an $$2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer,but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here,we show upregulated ST6Gal-I protein in several epithelial cancers,including many colon carcinomas. In normal colon,ST6Gal-I localized selectively to the base of crypts,where stem/progenitor cells are found,and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly,ST6Gal-I expression was restricted to basal epidermal layers in skin,another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells,with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations,we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells,as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely,short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively,our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations. View Publication

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules,we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization,lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization,dependent on dynein motor function,occurred before MTOC polarization,and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion,one that may prepare,but not commit cells for precision secretory function. View Publication

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with non-integrative constructs. Numerous studies,however,including those describing disease models,are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs,but in mesenchymal and endothelial iPSC derivatives,the transgenes experienced random up-regulation of Nanog and c-Myc. Additionally,we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies,which utilize cellular products derived from iPSCs generated with retro- or lentiviruses,should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work,however,is to communicate the possibility of transgene reactivation in retro- or lenti- iPSC derivatives and the associated loss of cellular fidelity in vitro,which may impact the outcomes of disease modeling and related experimentation. View Publication

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically,APL cells express PML-RAR,an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580,a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist,is a powerful inducer of granulocytic maturation in NB4,an APL-derived cell line,and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF),the compound induces granulocytic maturation,as assessed by determination of the levels of leukocyte alkaline phosphatase,CD11b,CD33,and G-CSF receptor mRNA,at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast,AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells,two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580,whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments,using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha,show that AM580 has a lower affinity than ATRA for both receptors. However,in the presence of PML-RAR,the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid,whereas,in the presence of RAR alpha,AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells. View Publication

T cell antigen-receptor (TCR) signaling controls the development,activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing,translation and stability of transcripts. In the present study,we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes,control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut ROR?t+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells. View Publication

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells,resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless,the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here,we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P {\textless} 0.00085),cleaved caspase (-3,-8,and -9) activity,and mitochondrial membrane potential reduction. Additionally,DAC-induced apoptosis was inhibited by Kp7-6,a FasL/Fas antagonist,indicating a crucial role of FasL/Fas,a cell death receptor,in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore,N-acetyl-L-cysteine (NAC),a widely accepted ROS scavenger,efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P {\textless} 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore,ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs,which serves as a crucial factor for mitochondrial membrane potential reduction,and DAC induces cell death prior to FasL/Fas stimulation. View Publication

BackgroundSchizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear,despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However,mechanistic insight at the protein level is limited.MethodsHere,we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach.ResultsSCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling,protein synthesis,cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control,SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore,we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells,indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ.ConclusionsThrough targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response,cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ,particularly during early development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12888-024-06127-x. View Publication

Despite recent advances in immunotherapies targeting single tumor-associated antigens,patients with multiple myeloma eventually relapse. ISB 2001 is a CD3+ T cell engager (TCE) co-targeting BCMA and CD38 designed to improve cytotoxicity against multiple myeloma. Targeting of two tumor-associated antigens by a single TCE resulted in superior cytotoxic potency across a variable range of BCMA and CD38 tumor expression profiles mimicking natural tumor heterogeneity,improved resistance to competing soluble factors and exhibited superior cytotoxic potency on patient-derived samples and in mouse models. Despite the broad expression of CD38 across human tissues,ISB 2001 demonstrated a reduced T cell activation profile in the absence of tumor cells when compared to TCEs targeting CD38 only. To determine an optimal first-in-human dose for the ongoing clinical trial (NCT05862012),we developed an innovative quantitative systems pharmacology model leveraging preclinical data,using a minimum pharmacologically active dose approach,therefore reducing patient exposure to subefficacious doses of therapies. Perro and colleagues develop a CD3+ T cell engager co-targeting BCMA and CD38 to improve immunotherapy for multiple myeloma,demonstrate cytotoxicity in patient-derived samples and murine models and develop a quantitative systems pharmacology model. View Publication

IntroductionCD8+ T cells are vital in the immune control of cancer and a key player in cell-based cancer immunotherapy. Recent studies have shown that microbial short-chain fatty acids (SCFA) can promote both effector and memory phenotypes in CD8+ T cells and may thereby enhance protection against cancer.MethodsIn this study,we determined the effect of SCFA butyrate on mouse CD8+ T cell function in vitro and in vivo,using the OT-I model.ResultsButyrate co-culture with anti-CD3 + anti-CD28 activated T cells in vitro enhanced the frequency of effector CD8+ IFN-γ-producing cells,and the amount of cytokine produced per cell. Culture with butyrate also enhanced the activation,TCR expression,and levels of phosphorylated mTOR proteins within CD8+ T cells but reduced proliferation rate and increased apoptosis. Butyrate-treated activated cells conferred tumor protection after adoptive transfer. Butyrate-treated cells were present at higher frequencies within the tumor compared to non-butyrate treated cells,and expressed IFN-γ. When analyzed using high dimensional cytometry,the tumors of mice that received butyrate-treated cells were enriched in clusters displaying an effector memory phenotype with high expression of IL-15Rβ and T-bet.DiscussionOur findings show that butyrate promotes the effector activity of CD8+ T cells in culture,which can persist in vivo while also stimulating memory phenotypes. Consequently,butyrate treatment may have strong application in T cell-based immunotherapies to improve protective cell functions and patient outcomes. View Publication

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients,respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (textgreater or = 98%) for CD73,CD105,CD106,CD29,CD13,CD44 and negative (textless or = 1%) for CD34,CD45,CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients,respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. View Publication