Overexpression of SOX4 in MSCs inhibits cellular senescence and enhances therapeutic efficacy in systemic lupus erythematosus

Abstract

Background Mesenchymal stem cells (MSCs) are widely used in treating autoimmune diseases. However, replicative senescence limits the quantity and quality of MSCs during population doublings in vitro. Transcription factor SOX4 is a crucial regulator of cell fate and stemness. This study aims to explore the role of SOX4 in senescence of MSCs and enhance their therapeutic efficacy in systemic lupus erythematosus (SLE). Methods In early-passage MSCs (P3), late-passage MSCs (P8), SOX4 downregulated P3-MSCs or SOX4 overexpressed P8-MSCs, cell morphology, mitochondrial reactive oxygen species (mtROS), senescence-associated β-galactosidase (SA-β-Gal) activity, cell proliferation rate, senescence-associated secretory phenotype (SASP) factors, cell cycle suppressors, the immunosuppressive effects on T cell activation and proliferation and the expression levels of SOX4 were determined. Imiquimod induced SLE mice were transplanted with P3-MSCs and P8-MSCs or control and SOX4 overexpressed P8-MSCs, and clinical symptoms were assessed. Results Compared to P3-MSCs, P8-MSCs display a senescent phenotype, increased mtROS, SA-β-Gal activity, SASP factors, and cell cycle suppressors p53, p21, and p16. Additionally, P8-MSCs have a reduced immunosuppressive function on T cell activation and proliferation, and express lower levels of SOX4. Downregulation of SOX4 in P3-MSCs promotes cellular senescence and impairs their immunosuppressive function. Conversely, overexpression of SOX4 in P8-MSCs ameliorates cellular senescence and enhances their immunosuppressive function. Furthermore, transplantation of P3-MSCs or SOX4-overexpressing P8-MSCs demonstrates greater therapeutic significantly efficacy in SLE mice compared to P8-MSCs.

1. Introduction

Mesenchymal stem cells (MSCs) are a highly heterogeneous cell type that can be easily isolated from various tissue sources, including bone marrow, umbilical cord, umbilical cord blood, placenta and adipose tissue [1]. They exhibit distinctive attributes, namely self-renewal capacity, pluripotency in terms of multilineage differentiation, and potent immunomodulatory effects. These attributes have positioned MSCs as viable candidates for therapeutic intervention and have been validated for their safety in clinical contexts concerning autoimmune disorders, with particular emphasis on systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [2, 3]. To achieve a sufficient cell yield for therapeutic applications, in vitro expansion of MSCs is necessary. Studies have documented that the age of donors negatively impacts the proliferative and migratory capabilities of MSCs, as well as leads to an upregulation in the expression of pro-inflammatory cytokines [4, 5]. Additionally, MSCs undergo replicative senescence as they are subjected to multiple population doublings in vitro, presenting a substantial barrier to their therapeutic applications [6].

Cellular senescence, a state characterized by irreversible cell-cycle arrest and the secretion of inflammatory cytokines, can occur in both normally proliferating cells and those that are impaired [7]. Reports indicate that stress-induced senescent MSCs express significantly high levels of pro-inflammatory factors, specifically interleukin (IL)-6 and IL-8 [8]. Late-passage MSCs inevitably show a senescent phenotype characterized by enlarged and flattened cell shapes, increased activity of senescence-associated β-galactosidase (SA-β-Gal), elevated expression of senescence-associated secretory phenotype (SASP)-related pro-inflammatory factors such as IL-1α and IL-8, and augmented production of ROS [9, 10]. Furthermore, late-passage MSCs demonstrate reduced proliferation and differentiation capabilities, decreased indoleamine-2,3-dioxygenase (IDO) activity, and diminished suppressive effects on pro-inflammatory T helper 1 (Th1) and Th17 cells when compared to early-passage MSCs [11–13]. However, the regulators and mechanisms that induce senescence in MSCs are not well understood.