Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.publishedVersio

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Abstract Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

SYNCHRONIZATION AND CHAOS IN THE DISCRETE CHEMICAL OSCILLATION SYSTEM(Session I : Cross-Disciplinary Physics, The 1st Tohwa University International Meeting on Statistical Physics Theories, Experiments and Computer Simulations)

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Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1β. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes