技术资料

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

OBJECTIVE True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS This was achieved by undertaking three sets of experiments. Firstly,SSEA-1+ and Oct-4??+??positive VSELs,sorted from GFP mice,were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly,uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly,OCT-4????expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS GFP??+??ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident,nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs,cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also,VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies. View Publication

The rare,accelerated aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is commonly caused by a de novo c.1824 C?>?T point mutation of the LMNA gene that results in the protein progerin. The primary cause of death is a heart attack or stroke arising from atherosclerosis. A characteristic feature of HGPS arteries is loss of smooth muscle cells. An adenine base editor (ABE7.10max) corrected the point mutation and produced significant improvement in HGPS mouse lifespan,vascular smooth muscle cell density,and adventitial fibrosis. To assess whether base editing correction of human HGPS tissue engineered blood vessels (TEBVs) prevents the HGPS vascular phenotype and to identify the minimum fraction of edited smooth muscle cells needed to effect such changes,we transduced HGPS iPSCs with lentivirus containing ABE7.10max. Endothelial cells (viECs) and smooth muscle cells (viSMCs) obtained by differentiation of edited HGPS iPSCs did not express progerin and had double-stranded DNA breaks and reactive oxygen species at the same levels as healthy viSMCs and viECs. Editing HGPSviECs restored a normal response to shear stress. Normal vasodilation and viSMC density were restored in TEBVs made with edited cells. When TEBVs were prepared with at least 50% edited smooth muscle cells,viSMC proliferation and myosin heavy chain levels significantly improved. Sequencing of TEBV cells after perfusion indicated an enrichment of edited cells after 5?weeks of perfusion when they comprised 50% of the initial number of cells in the TEBVs. Thus,base editing correction of a fraction of HGPS vascular cells improves human TEBV phenotype. View Publication

Increased proportions of CD8 T lymphocytes lacking expression of the CD28 costimulatory receptor have been documented during both aging and chronic infection with HIV-1,and their abundance correlates with numerous deleterious clinical outcomes. CD28-negative cells also arise in cell cultures of CD8(+)CD28(+) following multiple rounds of Ag-driven proliferation,reaching the end stage of replicative senescence. The present study investigates the role of a second T cell costimulatory receptor component,adenosine deaminase (ADA),on the process of replicative senescence. We had previously reported that CD28 signaling is required for optimal telomerase upregulation. In this study,we show that the CD8(+)CD28(+) T lymphocytes that are ADA(+) have significantly greater telomerase activity than those that do not express ADA and that ADA is progressively lost as cultures progress to senescence. Because ADA converts adenosine to inosine,cells lacking this enzyme might be subject to prolonged exposure to adenosine,which has immunosuppressive effects. Indeed,we show that chronic exposure of CD8 T lymphocytes to exogenous adenosine accelerates the process of replicative senescence,causing a reduction in overall proliferative potential,reduced telomerase activity,and blunted IL-2 gene transcription. The loss of CD28 expression was accelerated,in part due to adenosine-induced increases in constitutive caspase-3,known to act on the CD28 promoter. These findings provide the first evidence for a role of ADA in modulating the process of replicative senescence and suggest that strategies to enhance this enzyme may lead to novel therapeutic approaches for pathologies associated with increases in senescent CD8 T lymphocytes. View Publication

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages,adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine,but not those active on A1 (N6-(R)-phenylisopropyladenosine),A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine),or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors,induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact,both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover,the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1,a G1 cyclin-dependent kinase inhibitor,in a protein kinase A-dependent way. Moreover,the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor,the generation of cAMP,and the induction of p27kip-1 expression. View Publication

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations of methylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA),a metabolically stable analog of adenosine with high affinity for adenosine receptors,is an extremely potent behavioral depressant,reducing locomotor activity of mice at doses as little as 0.05 mumol/kg. The D isomer,which has much less affinity for adenosine receptors,is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine,when administered alone,elicits biphasic effects,with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA,even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However,like theophylline and caffeine,isobutylmethylxanthine reverses the L-PIA-evoked depression,converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors. View Publication

Spinobulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases,which are fatal neurodegenerative disorders mainly caused by protein-mediated toxic gain-of-function mechanisms and characterized by deposition of misfolded proteins in the form of aggregates. The neurotoxicity of the polyQ proteins can be modified by phosphorylation at specific sites,thereby providing the rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser(96) Phosphorylation of polyQ-AR by CDK2 increased protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy,we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP),a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser(96) phosphorylation,promoted polyQ-AR degradation,and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA. View Publication

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs),partially reprogrammed cells,somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over approximately 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free,enriched to 95%-99% purity with textgreater80% survival,and had normal transcriptional profiles,differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells. View Publication

Background and Aims Disturbance of intestinal homeostasis is associated with the development of inflammatory bowel disease (IBD),and TGF-beta$ signaling impairment in mononuclear phagocytes (MPs) causes murine colitis with goblet cell depletion. Here,we examined an organoid-MP co-culture system to study the role of MPs in intestinal epithelial differentiation and homeostasis. Methods Intestinal organoids were co-cultured with lamina propria leukocytes and bone marrow-derived dendritic cells (BMDCs) from CD11c-cre Tgfbr2fl/fl mice. Organoid-MP adhesive interactions were evaluated by microscopy,RT-PCR,and flow cytometry. Murine colitis models (dextran sodium sulphate (DSS),CD11c-cre Tgfbr2fl/fl,T-cell-transfer) were used for histological and immunohistochemical analysis. Anti-E-cadherin antibody treatment or CD11c+-cell-specific CDH1 gene deletion were performed for E-cadherin neutralization or knockout. Colonic biopsies from patients with ulcerative colitis were analyzed by flow cytometry. Results Intestinal organoids co-cultured with CD11c+ lamina propria leukocytes or BMDCs from CD11c-cre Tgfbr2fl/fl mice showed morphological changes and goblet cell depletion with Notch signal activation,analogous to CD11c-cre Tgfbr2fl/fl colitis. E-cadherin was upregulated in CD11c+ MPs,especially CX3CR1+CCR2+ monocytes,of CD11c-cre Tgfbr2fl/fl mice. E-cadherin-mediated BMDC adhesion promoted Notch activation and cystic changes in organoids. Anti-E-cadherin antibody treatment attenuated colitis in CD11c-cre Tgfbr2fl/fl and T-cell-transferred mice. In addition,E-cadherin deletion in CD11c+ cells attenuated colitis in both CD11c-cre Tgfbr2fl/fl and DSS-treated mice. In patients with ulcerative colitis,E-cadherin expressed by intestinal CD11c+ leukocytes was enhanced compared with that in healthy controls. Conclusions E-cadherin-mediated MP-epithelium adhesion is associated with the development of colitis,and blocking these adhesions may have therapeutic potential for IBD. View Publication

AbstractBackgroundThe tumor microenvironment (TME) poses challenges that limit the efficacy of conventional CAR-T cell therapies. Homing barriers,immunosuppressive factors,and target antigen heterogeneity can impair CAR-T cell functional activity within the TME. Alternative strategies have contemplated incorporating the use of gamma delta (γδ) T cells as a CAR-T cell approach to potentially overcome these limitations. γδ T cells possess both innate and adaptive immunity to facilitate broad tumor recognition,and their natural propensity for tissue tropism may allow for more effective tumor infiltration. Reported here is the preclinical characterization of ADI-270,an allogeneic γδ CAR-T cell product targeting CD70+ cancers,engineered with a third-generation CAR based on the natural CD27 receptor. ADI-270 is also double-armored to mitigate the immunosuppressive effects of TGFβ and reduce the potential for allogeneic rejection.MethodsVδ1 T cells engineered to express an anti-CD70 CAR and dominant negative TGFβ receptor II (dnTGFβRII) were expanded from healthy donor human PBMCs. The phenotype and functional characterization of ADI-270 were assessed with in vitro cell culture assays and in vivo tumor xenograft models.ResultsADI-270 exhibited high levels of in vitro cytotoxicity against a panel of cancer cell lines and displayed a favorable inflammatory cytokine profile compared with reference scFv-based anti-CD70 CAR αβ T cells. Cytotoxicity remained potent despite low CD70 expression observed in multiple solid and hematologic tumor cell models. When armored with dnTGFβRII,ADI-270 exhibited functional resilience to TGFβ-mediated inhibition of T cell effector activity. In addition,the incorporation of potent and sensitive CD70-targeting decreased T cell-mediated alloreactive killing against ADI-270 in vitro without evidence of fratricide. Finally,ADI-270 displayed robust tumor tropism and control of primary and secondary tumor challenges in xenograft mouse models.ConclusionsThese results demonstrate the robust potency and capacity of ADI-270 to extend antitumor activity to cancers with heterogeneous antigen expression. The functional armoring incorporated into ADI-270 provides a mechanism to overcome the limitations of reduced efficacy and persistence within the TME. ADI-270 has the potential to target multiple CD70+ cancers with initial clinical evaluation proceeding in relapsed/refractory clear cell renal cell carcinoma.Trial registration numberNCT06480565. View Publication

Interactions between adipose tissue,liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model,we establish an interconnected microphysiological system (MPS) containing white adipocytes,hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS,we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function,whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance,corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases. In vitro modelling of the adipose tissue-liver axis can advance understanding and therapy of metabolic disease,including by distinguishing effects of obesity and inflammation. Here,authors develop such a system based on isogenic human iPSCs and interconnected microphysiological devices. View Publication