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

Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study,a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline,MCF-7 AdVp3000. Levels were lower in earlier steps of selection,and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells,but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter,which is overexpressed in mitoxantrone-resistant cells. View Publication

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO),a chemical modulator of small-/intermediate-conductance Ca(2+)-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs),we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs,timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs,including an increase in nodal- and atrial-like phenotypes. However,our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and,subsequently,CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO,presumably via an SK-independent mechanism. Together,EBIO did not promote cardiogenic differentiation of PSCs,opposing previous findings,but triggered lineage-selective survival at a cardiac progenitor stage,which we propose as a pharmacological strategy to modulate CM subtype composition. View Publication

Lateral Meningocele Syndrome (LMS),a disorder associated with NOTCH3 pathogenic variants,presents with neurological,craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs,induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs,NOTCH36692-93insC and isogenic controls,free of chromosomal aberrations as determined by human CytoSNP850 array,were cultured under conditions of neural crest,mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1,HEY1,2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL,BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70–80% in mesenchymal cells and by 45–55% in osteogenic cells and NOTCH3 mRNA by 15–30% and 20–40%,respectively. In conclusion,a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS. View Publication

SummaryAlthough chimeric antigen receptor (CAR) T cells have demonstrated therapeutic activity in hematopoietic malignancies,tumor heterogeneity has impeded the efficacy of CAR T cells and their extension into successful solid tumor treatment. To address these challenges,induced pluripotent stem cell (iPSC)-derived T (iT) cells are engineered to uniformly express CAR and T cell receptor (TCR),enabling targeting of both surface and intracellular antigens,respectively,along with a high-affinity,non-cleavable variant of CD16a (hnCD16) to support antibody-dependent cellular cytotoxicity (ADCC) when combined with therapeutic antibodies. Co-expression of each antitumor strategy on engineered iT cells enables independent and antigen-specific targeting across a diverse set of liquid and solid tumors. In heterogeneous tumor models,coactivation of these modalities is required for measurable antitumor efficacy,with activation of all three modalities displaying maximal efficacy. These data highlight the therapeutic potential of an off-the-shelf engineered iPSC-derived trimodal T cell expressing CAR,TCR,and hnCD16 to combat difficult-to-treat heterogeneous tumors. Graphical abstract Highlights•CAR,TCR,and hnCD16 can be uniformly co-expressed and can function in iT cells•hnCD16 signals through CD3ζ and arms iT cells with targeting flexibility through ADCC•Concurring CAR,TCR,and hnCD16 activation demonstrates a cooperative effect•Multi-targeting with trimodal iT cells can control heterogeneous tumors in vivo Yang et al. show that (1) trimodal iPSC cells expressing CAR,TCR,and hnCD16 can commit to T cell lineage,(2) hnCD16 signals through CD3ζ in iT cells and arms iT cells with ADCC targeting flexibility,and (3) trimodal iT cells control antigen-heterogeneous tumors in vivo through multi-modal targeting. View Publication

Respiratory syncytial virus (RSV) infection in the upper respiratory tract promotes disease progression and transmission,with excessive inflammation contributing to severe lower respiratory tract involvement. This study investigates the immunomodulatory effects of Lactobacillus rhamnosus D3189 on viral kinetics and innate immune responses in well-differentiated nasal epithelial cells (WD-NECs). WD-NECs from healthy adult donors (N = 8) were cultured in vitro,treated with L. rhamnosus D3189,and then infected with RSV (strain RS4) 24 hours later. Viral replication and shedding were assessed via RT-qPCR and plaque assays. Cytotoxicity and epithelial integrity were evaluated using LDH release and transepithelial electrical resistance (TEER). Inflammatory and antiviral responses were investigated using multiplex immunoassays,AlphaLISA,and ELISA. RSV infection induced robust viral replication and shedding,disrupted epithelial barrier integrity,and triggered the release of pro-inflammatory cytokines and type I/III interferons. L. rhamnosus D3189 alone did not induce cytotoxicity or inflammation. While it had no effect on viral replication,TEER,LDH release,or IFN-λ1/3 levels,D3189 significantly enhanced IFN-β production,reduced viral shedding,and attenuated RSV-induced cytokine and chemokine responses. L. rhamnosus D3189 modulates the epithelial immune response to RSV,reducing inflammation and viral shedding without compromising epithelial integrity. These findings support its potential as a novel strategy to limit RSV-associated infection and transmission. View Publication

Strategies targeting leukemic stem and progenitor cells (LSPCs) are needed for durable remissions in acute myeloid leukemia (AML) and high-risk myelodysplastic neoplasms (MDS). While CD123 constitutes a promising target on LSPCs and leukemic blasts,previous CD123-targeting approaches showed limited efficacy and challenging safety profiles. Here,we describe the preclinical efficacy and safety of the bispecific CD123/CD16A innate cell engager “AFM28”,demonstrating superior activity against AML and MDS patient-derived LSPCs and blasts in vitro compared to an Fc-enhanced CD123-targeting antibody,especially towards CD123 low and/or CD64 + leukemic cells. AFM28 induces autologous anti-leukemic activity in fresh AML whole blood cultures,demonstrating its potential to enhance NK cell function from AML patients. Responsiveness can be further enhanced by allogeneic NK cell addition. Anti-leukemic activity of AFM28 is confirmed in xenograft mouse models. In addition,AFM28 is well tolerated and demonstrates pharmacodynamic activity in cynomolgus monkeys. Altogether,our results indicate that AFM28 has the potential to reduce relapse-inducing residual disease and promote long-term remissions for patients with AML and MDS with a favorable safety profile. Subject terms: Cancer immunotherapy,Preclinical research,Acute myeloid leukaemia,Myelodysplastic syndrome View Publication

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here,we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead,such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction,and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus,FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells,which fail to undergo either neural or mesodermal differentiation in adherent culture,and retain expression of pluripotency markers Oct4,Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos. View Publication

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells,we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model,donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly,highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer,accumulated in vivo at advanced proliferative cycles,and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted,apoptosis-resistant phenotype,including: 1) reduced apoptosis after staurosporine addition,serum starvation,or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection,we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4,IL-10,perforin,or Fas ligand; 2) could not be reversed by IL-2,IL-7,or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis,persist in vivo,and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells. View Publication

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems,we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas,high ALDH activity identifies the tumorigenic cell fraction,capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas,expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts. View Publication

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study,we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover,reactive oxygen species as well as activated caspase 3,8,and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly,increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2,GD2,and GD3 play a significant role in immune dysfunction observed in RCC patient T cells. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication