The objective of the study is to establish an in vitro model using iPSCs to test the hypotheses developed.
The primary objective is to generate, via the SAFE-IPS platform, 8 iPSC lines derived from blood samples taken from:
* Two patients with severe/diffuse SSc with multi-organ involvement (with anti-SCl-70 autoantibodies) * Two of their healthy close relatives * Two patients with uncomplicated SSc with localized involvement (with non-SCl-70 autoantibodies) * Two of their healthy close relatives
These 8 cell lines will be differentiated by several teams at the FHU REGENHAB into:
* Immune cells (monocytes/macrophages, neutrophils, dendritic cells, B/T lymphocytes) * Mesenchymal stromal cells * Myocardial cells * Skin cells (fibroblasts) * Synovial cells * Bronchial cells * Endothelial cells
This project aims to map cellular and tissue heterogeneity using iPSC lines obtained from patients with both severe and mild SSc, employing single-cell RNA-seq under various pathological conditions, with and without autologous (or even heterologous) autoimmune stimulation. For example, the percentages of different cell types comprising a tissue in these various situations will be calculated. Comparisons will be made with control groups using healthy iPSC lines by recruiting healthy subjects with the same genetic profile and gender as the patients.
Age between 18 and 85 years
Diagnosis of diffuse/severe systemic sclerosis by a physician for at least 12 months based on recommendations (according to ACR criteria)
Known positivity of autoimmunity directed against Scl-70
Diagnosis of systemic sclerosis by a physician for at least 12 months based on recommendations (according to ACR criteria)
Other diseases that may affect erythroid progenitor cells (non-exhaustive: active cancer, hematological malignancy, chemotherapy targeting DNA)
Patient in an exclusion period determined by another protocol
Protected populations according to French public health law (pregnant or breastfeeding women; individuals deprived of liberty by judicial or administrative decision; adults under legal protection (any form of guardianship))
Absence of informed consent
Single group
Blood Sample Collection
Procedure/SurgeryCollection of approximately 28 mL of blood from each participant to generate induced pluripotent stem cell (iPSC) lines for in vitro cellular and tissue modeling analyses.
Rate of successful iPSC line generation from PBMCs in diffuse/severe systemic sclerosis patients
Percentage of participants with diffuse/severe systemic sclerosis (anti-Scl-70 positive) from whom at least one iPSC line is successfully generated via Sendai virus reprogramming of peripheral blood mononuclear cells (PBMCs). Unit of Measure: % of participants with at least one validated iPSC line
Rate of successful differentiation of iPSCs into at least one target functional cell type in localized/non-complicated systemic sclerosis patients
Among validated iPSC lines from localized/non-complicated SSc patients, the percentage of lines achieving successful directed differentiation into at least one target cell type (cardiomyocytes, macrophages, bronchial epithelial cells, immune cells, or fibroblasts). Success is confirmed by cell-type-specific marker expression assessed by immunofluorescence and flow cytometry. Unit of Measure: % of validated iPSC lines successfully differentiated into ≥1 target cell type
Rate of successful iPSC line generation from PBMCs in localized/non-complicated systemic sclerosis patients
Percentage of participants with localized/non-complicated systemic sclerosis (anti-Scl-70 negative, other SSc-specific antibody positive) from whom at least one iPSC line is successfully generated via Sendai virus reprogramming of PBMCs. Unit of Measure: % of participants with at least one validated iPSC line
Rate of successful differentiation of iPSCs into at least one target functional cell type in diffuse/severe systemic sclerosis patients
Among validated iPSC lines from diffuse/severe SSc patients, the percentage of lines achieving successful directed differentiation into at least one target cell type (cardiomyocytes, macrophages, bronchial epithelial cells, immune cells, or fibroblasts). Success is confirmed by cell-type-specific marker expression assessed by immunofluorescence and flow cytometry (e.g., cTnT for cardiomyocytes; CD68/CSFR1 for macrophages; NKX2.1/MUC5AC for bronchial epithelium). Unit of Measure: % of validated iPSC lines successfully differentiated into ≥1 target cell type
Change in cardiomyocyte differentiation efficiency following autologous autoantibody exposure
Comparison of ventricular cardiomyocyte differentiation efficiency, expressed as percentage of cardiac troponin T-positive (cTnT+) cells by flow cytometry, in iPSC-derived cardiomyocytes cultured with vs. without addition of autologous serum containing patient-specific autoantibodies. Serum is collected at Day 0 blood draw and stored at -80°C until use. Unit of Measure: % cTnT-positive cells (flow cytometry) - with vs. without autologous serum
Change in macrophage differentiation and inflammatory cytokine secretion following autologous autoantibody exposure
Assessment of iPSC-derived macrophage phenotype (% CD68+, CSFR1+, CD163+ cells by flow cytometry) and inflammatory response (secretion of CCL2, IL-6, CXCL1, CXCL8 measured by ELISA in pg/mL) under baseline conditions and following addition of autologous autoantibody-containing serum, compared between SSc patient-derived and healthy control-derived macrophages. Unit of Measure: Cytokine concentration (pg/mL) by ELISA; % CD68+/CSFR1+ cells by flow cytometry
Functional characterization of iPSC-derived cardiomyocytes - spontaneous beating activity
Proportion of iPSC-derived ventricular cardiomyocyte clusters exhibiting spontaneous contractile activity, assessed by video microscopy, as a measure of functional maturation following 2D differentiation and 3D spheroid formation using Matrigel. Maturation is further assessed after small molecule treatment with triiodothyronine (T3) and lipid-soluble cyclic AMP. Unit of Measure: % spontaneously beating clusters (video microscopy)
Molecular characterization of iPSC-derived cardiomyocytes - cardiac marker expression
Assessment of cardiomyocyte identity by flow cytometry (% cardiac troponin T-positive cells) and RT-qPCR quantification of cardiac-specific genes (MYH6, MYH7, TNNI3, KCNH2) in iPSC-derived cardiomyocytes from SSc patients compared to healthy matched controls. Unit of Measure: % cTnT-positive cells (flow cytometry); relative gene expression (RT-qPCR, arbitrary units normalized to housekeeping gene)
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