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

Disease progression and drug resistance in patients with chronic lymphocytic leukaemia (CLL) depend on signals from the tumour microenvironment in lymphoid sites. GRK2 inhibits the egress of normal B cells from lymphoid tissues by inducing the downregulation of the S1P-receptor 1 (S1PR1). In this study we investigated the role of GRK2 in the context of CLL using in vitro and in vivo murine models,and also primary samples from CLL patients. We found that pharmacological inhibition of GRK2 enhanced the migration of leukemic cells from CLL patients towards S1P and impaired the S1P-induced downregulation of S1PR1. Likewise,CRISPR/Cas9-mediated GRK2 deletion in a murine leukemic cell line derived from the Eµ-TCL1 mouse model of CLL also increased migratory capacity toward S1P in vitro. Furthermore,when injected into mice,GRK2-deficient murine leukemic cells exhibited an altered in vivo localization,with a higher presence in the blood and spleen compared to the bone marrow. Within the spleen,these cells displayed reduced localization to the follicles compared to control murine leukemic cells. Deletion of GRK2 on murine leukemic cells did not affect their in vitro proliferation,but notably,conferred a growth disadvantage in vivo. These findings underscore GRK2 as a critical regulator of the localization of CLL cells in vivo and suggest its potential as a therapeutic target to disrupt survival niches in CLL.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-91536-5. View Publication

BRAF-activating mutations have been reported in several types of cancer,including melanoma ( approximately 70% of cases),thyroid (30-70%),ovarian (15-30%),and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts,indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375,the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing,with little or no cell death. Reexpression of the thyroid differentiation marker,sodium iodide symporter,was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor,PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether,our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore,we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. View Publication

A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs),including professional antigen-presenting cells such as dendritic cells (DCs),play a central role in T-cell biology. Here,we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell-derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes,but cell death was significantly increased after monocyte depletion. Furthermore,monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin,and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES),where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively,the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders. View Publication

A subset of cells,tentatively called cancer stem cells (CSCs),in breast cancer have been associated with tumor initiation,drug resistance,and tumor persistence or aggressiveness. They are characterized by CD44 positivity,CD24 negativity,and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed,flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue,with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years,we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1,CD44,and cytokeratin to measure the coexpression of these proteins,putative CSCs appear in variable sized clusters and in 27 cases (of 490),which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size,histological grade,nodal status,ER-,PR,- and HER2-status. In this cohort,ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however,the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously,we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover,the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here,we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways,which are recognized as drivers of resistance. Mechanistically,this drug combination attenuated both interacting signaling networks,leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently,leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK,JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation,viability and the stress response. Collectively,these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment. View Publication

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however,CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs,leading to disease relapse. To prevent this,new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34 + CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore,the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C 33 H 61 NO 5 S) is a structurally simplified analog of JCI-20679,the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here,we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34 + CML cells isolated from the bone marrow of untreated CML patients. Taken together,these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs,and may improve the treatment of CML. View Publication

Hypertrophic cardiomyopathy (HCM) is an inherited cardiomyopathy characterized by left ventricular hypertrophy ≥15 mm in the absence of loading conditions. HCM has a prevalence of up to one in 200,and can result in significant adverse outcomes including heart failure and sudden cardiac death. An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells obtained from the whole blood of a 38-year-old female patient with HCM in which genetic testing identified the well-known pathogenic p.Arg403Gln mutation in myosin heavy chain 7. iPSCs express pluripotency markers,demonstrate trilineage differentiation capacity,and display a normal 46,XX female karyotype. This resource will allow further assessment of the pathophysiological development of HCM. View Publication

Background: Takotsubo cardiomyopathy (TTC) is marked by an acute,transient,and reversible left ventricular systolic dysfunction triggered by stress,with endothelial dysfunction being one of its pathophysiological mechanisms. However,the precise molecular mechanism underlying the interaction between endothelial cells and cardiomyocytes during TTC remains unclear. This study reveals that exosomal miRNAs derived from endothelial cells exposed to catecholamine contribute to ion channel dysfunction in the setting of TTC. Methods: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with epinephrine (Epi) or exosomes (Exo) from Epi-treated human cardiac microvascular endothelial cells (HCMECs) or Exo derived from HCMECs transfected with miR-126-3p. The immunofluorescence staining,flow cytometry,qPCR,single-cell contraction,intracellular calcium transients,patch-clamp,dual luciferase reporter assay and western blot were performed for the study. Results: Modeling TTC with high doses of epinephrine (Epi) treatment in hiPSC-CMs shows suppression of depolarization velocity (Vmax),prolongation of action potential duration (APD),and induction of arrhythmic events. Exo derived from HCMECs treated with Epi (Epi-exo) mimicked or enhanced the effects of Epi. Epi exposure led to elevated levels of miR-126-3p in both HCMECs and their exosomes. Exo enriched with miR-126-3p demonstrated similar effects as Epi-exo,establishing the crucial role of miR-126-3p in the mechanism of Epi-exo. Dual luciferase reporter assay coupled with gene mutation techniques identified that miR-126-3p was found to target the regulator of G-protein signaling 3 (RGS3) gene. Western blot and qPCR analyses confirmed that miR-126-3p-mimic reduced RGS3 expression in both HCMECs and hiPSC-CMs,indicating miR-126-3p inhibits RGS3 signaling. Additionally,miR-126-3p levels were significantly higher in the serum of TTC patients compared to healthy controls and patients who had recovered from TTC. Conclusions: Our study is the first to reveal that exosomal miR-126-3p,originating from endothelial cells,contributes to ion channel dysfunction by regulating RGS3 signaling in cardiomyocytes. These findings provide new perspectives on the pathogenesis of TTC and suggest potential therapeutic targets for treatment. View Publication

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow-derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice,but none of the untreated controls,survived for textgreater or = 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD,although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low,but surviving Purkinje cells contained the retrovirally expressed human enzyme,and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow-derived MSCs. View Publication

Although significant advances have been made over the last decade with respect to our understanding of stem cell biology,progress has been limited in the development of successful techniques for clinically significant ex vivo expansion of hematopoietic stem and progenitor cells. We here describe the effect of Notch ligand density on induction of Notch signaling and subsequent cell fate of human CD34+CD38- cord blood progenitors. Lower densities of Delta1(ext-IgG) enhanced the generation of CD34+ cells as well as CD14+ and CD7+ cells,consistent with early myeloid and lymphoid differentiation,respectively. However,culture with increased amounts of Delta1(ext-IgG) induced apoptosis of CD34+ precursors resulting in decreased cell numbers,without affecting generation of CD7+ cells. RNA interference studies revealed that the promotion of lymphoid differentiation was primarily mediated by Delta1 activation of Notch1. Furthermore,enhanced generation of NOD/SCID repopulating cells was seen following culture with lower but not higher densities of ligand. These studies indicate critical,quantitative aspects of Notch signaling in affecting hematopoietic precursor cell-fate outcomes and suggest that density of Notch ligands in different organ systems may be an important determinant in regulating cell-fate outcomes. Moreover,these findings contribute to the development of methodology for manipulation of hematopoietic precursors for therapeutic purposes. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication