搜索结果: 'gmp t cell activator'

Abstract Purpose: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic,with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells,but previous studies have shown variability in iPSC propensity to differentiate into RPE. Methods: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid,directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE,which were characterized with respect to global gene expression,expression of RPE markers,and cellular function. Results: We found that all 5 iPSC lines (iPSC-1,iPSC-2,iPSC-3,iPSC-4,and iPSC-12) generated RPE using the directed differentiation protocol; however,2 of the 5 iPSC lines (iPSC-4 and iPSC-... View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts,which eventually blocks signal propagation and often induces receptor internalization. However,there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2,GRK3,GRK5,GRK6) simultaneously,but we have very limited knowledge about their interplay in primary immune cells. In particular,we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type,and (b) one specific GPCR between several immune cell subsets. To address this issue,we generated mouse models of single,combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils,T cells,B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes,many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands,whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types,and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly,we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis,which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together,our findings demonstrate the complexity of GRK functions in immune cells,which go beyond GPCR desensitization in specific leukocyte types. Furthermore,they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset. View Publication

Cell replacement therapy is a promising therapeutic option for dry age-related macular degeneration (AMD). In this study,we outline our design for scalable manufacture with appropriate quality gates and present in vivo data for establishing preclinical safety and efficacy of an induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) product,thus laying the foundation for Phase 1/2a trial approval in India (ClinicalTrials.gov ID: NCT06394232; date of registration: 23rd September 2024). Escalating doses of RPE cell suspension in immunocompromised animals demonstrated absence of tumor formation up to 9?months post-injection. Good Laboratory Practices (GLP) toxicology and tolerability studies in rabbits and non-human primates (NHP) respectively showed no major adverse events. RPE transplanted into immune suppressed RCS rats showed integration,neuroprotection and rescue of visual function. In addition,we provide a detailed description of the modifications in GMP manufacturing protocol to create a final product with a unique composition and Chemistry,Manufacturing and Controls (CMC) studies performed during product development. View Publication

Despite extensive research on microbiome alterations in ulcerative colitis (UC),the role of the constituent stable microbiota remains unclear. This study,employing 16S rRNA-gene sequencing,uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis,the study highlights microbial constituents,featuring Phocaeicola,Collinsella,Roseburia,Holdemanella,and Bacteroides,that are not affected during the course of UC. Co-cultivation experiments,utilizing commensal Escherichia coli and Phocaeicola vulgatus,were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells,without inducing pathogen recognition and stress responses,offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients’ response to the same bacteria,which indicate the need for more comprehensive,stratified analyses with an expanded sample size. This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota – Escherichia coli and Phocaeicola vulgatus – potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals. The online version contains supplementary material available at 10.1186/s13099-024-00612-0. View Publication

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease,and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed,and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells,we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions,including irregularly enlarged cell morphology,cell cycle arrest with downregulation of Ki67,and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover,immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly,HPV surrogate marker p16,and stress-associated basal keratinocyte marker KRT17,indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation,and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable. Subject terms: Oral cancer,Mechanisms of disease,Cell death View Publication

Tumor-initiating cells (TICs) share features and regulatory pathways with normal stem cells,yet how the stem cell niche contributes to tumorigenesis remains unclear. Here,we identify CXCR4 + macrophages as a niche population enriched in normal mammary ducts,where they promote the regenerative activity of basal cells in response to luminal cell-derived CXCL12. CXCL12 triggers AKT-mediated stabilization of β-catenin,which induces Wnt ligands and pro-migratory genes,enabling intraductal macrophage infiltration and supporting regenerative activity of basal cells. Notably,these same CXCR4 + niche macrophages regulate the tumor-initiating activity of various breast cancer subtypes by enhancing TIC survival and tumor-forming capacity,while promoting early immune evasion through regulatory T cell induction. Furthermore,a CXCR4 + niche macrophage gene signature correlates with poor prognosis in human breast cancer. These findings highlight the pivotal role of the CXCL12-CXCR4 axis in orchestrating interactions between niche macrophages,mammary epithelial cells,and immune cells,thereby establishing a supportive niche for both normal tissue regeneration and mammary tumor initiation. Subject terms: Cancer stem cells,Cancer microenvironment,Tumour immunology View Publication

Human airways contain specialized rare epithelial cells including CFTR-rich ionocytes that regulate airway surface physiology and chemosensory tuft cells that produce asthma-associated inflammatory mediators. Here,using a lung cell atlas of 311,748 single cell RNA-Seq profiles,we identify 687 ionocytes (0.45%). In contrast to prior reports claiming a lack of ionocytes in the small airways,we demonstrate that ionocytes are present in small and large airways in similar proportions. Surprisingly,we find only 3 mature tuft cells (0.002%),and demonstrate that previously annotated tuft-like cells are instead highly replicative progenitor cells. These tuft-ionocyte progenitor (TIP) cells produce ionocytes as a default lineage. However,Type 2 and Type 17 cytokines divert TIP cell lineage in vitro,resulting in the production of mature tuft cells at the expense of ionocyte differentiation. Our dataset thus provides an updated understanding of airway rare cell composition,and further suggests that clinically relevant cytokines may skew the composition of disease-relevant rare cells. Subject terms: Interleukins,Systems analysis,Differentiation,Sequencing View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 72-year old female Parkinson's disease (PD) patient with R1398H variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model provides a good platform for studying the mechanism of PD,and also for drug testing and gene therapy studies. View Publication

Skeletal muscle aging results in a gradual loss of skeletal muscle mass,skeletal muscle function and regenerative capacity,which can lead to sarcopenia and increased mortality. Although the mechanisms underlying sarcopenia remain unclear,the skeletal muscle stem cell,or satellite cell,is required for muscle regeneration. Therefore,identification of signaling pathways affecting satellite cell function during aging may provide insights into therapeutic targets for combating sarcopenia. Here,we show that a cell-autonomous loss in self-renewal occurs via alterations in fibroblast growth factor receptor-1,p38α and p38β mitogen-activated protein kinase signaling in satellite cells from aged mice. We further demonstrate that pharmacological manipulation of these pathways can ameliorate age-associated self-renewal defects. Thus,our data highlight an age-associated deregulation of a satellite cell homeostatic network and reveal potential therapeutic opportunities for the treatment of progressive muscle wasting. View Publication

Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor,the aryl hydrocarbon receptor (AHR),has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AHR plays a role in both the acquisition and progression of OSCC,as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis,AHR activity in two oral squamous cell lines was modulated with AHR prototypic,environmental and bacterial AHR ligands,AHR-specific inhibitors,and phenotypic,genomic and functional characteristics were evaluated. The data demonstrate that: 1) primary OSCC tissue expresses elevated levels of nuclear AHR as compared to normal tissue,2) Ahr mRNA expression is up-regulated in 320 primary OSCC,3) AHR hyper-activation with several ligands,including environmental and bacterial ligands,significantly increases AHR activity,ALDH1 activity,and accelerates cell migration,4) AHR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance,5) AHR knockdown inhibits tumorsphere formation in low adherence conditions,and 6) AHR knockdown inhibits tumor growth and increases overall survival in vivo. These data demonstrate that the AHR plays an important role in development and progression of OSCC,and specifically cancer stem-like cells. Prototypic,environmental and bacterial AHR ligands may exacerbate OSCC by enhancing expression of these properties. IMPLICATIONS This study,for the first time,demonstrates the ability of diverse AHR ligands to regulate AHR activity in oral squamous cell carcinoma cells,as well as regulate several important characteristics of oral cancer stem cells,in vivo and in vitro. View Publication

Bone marrow progenitor cells develop into mature tissue myeloid cells under the influence of colony-stimulating factors. Cytokines that are elevated post-thermal injury have been shown to influence this process. We hypothesize that thermal injury alters myelopoiesis at the level of the progenitor cell. These differences should be visible after in vitro cultures that include colony-stimulating factors. Prior to culture,bone marrow at postburn day 1 (PBD1) was assessed for cell surface markers and the levels of myeloid progenitors. After culture in granulocyte/macrophage-stimulating colony-stimulating factor,the cell surface markers of the cultured cells were determined. PBD1 marrow from thermally injured rats had more progenitor cells responsive to granulocyte/macrophage-stimulating colony-stimulating factor than did sham. Cultured PBD1 marrow produced more CD90(br) MY(br) CD45(dim) CD4(-) MHCII(-) CD11b(dim) eosinophils than did sham. Cultured bone marrow from thermally injured animals produces myeloid cells with an altered phenotype. Similar changes in myelopoiesis may take place in vivo. View Publication