Tissue Modeling in Systemic Sclerosis Using Induced Pluripotent Stem Cells (iPSCs)

Background:

Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease that predominantly affects women, often has a severe course, and is characterized by heterogeneous multiorgan involvement. Its pathophysiology, involving microvascular abnormalities, autoimmune activation, and progressive fibrosis, remains incompletely understood, particularly regarding its variations across clinical forms and affected organs. Currently available treatments rely primarily on immunosuppressive strategies, which have limited efficacy and are associated with significant adverse effects, with no therapeutic option available to prevent certain major complications. In this context of unmet medical need, the use of induced pluripotent stem cells (iPSCs) derived from patients with SSc represents an innovative approach to modeling the pathophysiological mechanisms of the disease in vitro. The FHU Regenhab consortium's demonstrated ability to differentiate iPSCs into several relevant cell types offers a unique opportunity to study specific organ damage and identify new therapeutic targets, paving the way for more personalized treatment strategies.

Objectives:

Primary objective:

The primary objective is to generate, using the SAFE-IPS platform, 8 iPSC lines derived from cells obtained from a blood sample from:

• Two patients with severe/diffuse SSc and 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 by recruiting patients with severe and mild SSc, via 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.

Secondary objectives:

Signaling pathways and major biological processes that are differentially activated and suppressed under various conditions will be analyzed comparatively to better understand the extent to which the addition of the autoantibody alters cellular biology.

The identified signaling pathways will enable pharmacological modulation of differentiated tissue cultures. For example: if the ERK2/3 pathway is more active in differentiated tissues obtained from SSc patients compared to controls, as reported in Kim's publication (SCRT 2022), pharmacological modulation will be tested with and without the addition of autoantibodies. The tissue phenotype will be recharacterized using immunohistochemistry, conventional biochemistry for protein expression, and transcriptomic analysis.

Study Population:

A maximum of 8 patients will be enrolled, with the aim of having at least two evaluable patients per group: two patients with severe/diffuse SSc and two patients with localized/uncomplicated SSc. Enrollment will be halted once two iPSC lines differentiated into at least one target cell type have been obtained for each condition.

Investigators will simultaneously include healthy subjects without autoimmune diseases matched to each patient (n=8 maximum).

Inclusion criteria:

• Age between 18 and 85 years
• Participant agrees to have their biological samples and cell lines stored in a biological sample collection for other research purposes (including genetic research) on scleroderma

Severe SSc group:

• Diagnosis of diffuse/severe SSc by a physician for at least 12 months based on guidelines (according to ACR criteria)
• With known positivity for Scl-70 autoantibodies.

Localized/Uncomplicated SSc Group

• Diagnosis of SSc by a physician for at least 12 months based on guidelines (according to ACR criteria)
• Documentation of the absence of major involvement of vital organs
• Negative anti-Scl-70 autoantibody but positive for other SSc Dot antibodies

Healthy Subjects Group

• Relative of a patient enrolled in one of the scleroderma groups (father, mother, brother, sister, adult child)
• Absence of systemic autoimmune diseases

Exclusion criteria:

• Other diseases that may affect erythroid progenitor cells (non-exhaustive list: active cancer, malignant hematological disease, DNA-targeted chemotherapy)
• Patient in an exclusion period determined by another protocol
• Protected populations as defined by the French Public Health Code (women who are giving birth, breastfeeding, or pregnant; individuals deprived of their liberty by judicial or administrative decision; adults under legal protection (under any form of guardianship))
• Lack of informed consent
• Not covered by the national health insurance system