Deletion of p18INK4c enhances both osteogenesis and hematopoietic supportive capacity of bone marrow mesenchymal stromal cells.

Abstract

Background: p18^{INK4 C} (CDKN2C, encoded by p18^INK4c or Cdkn2c) is an early G1-phase cyclin-dependent kinase inhibitor protein. Previous studies demonstrated enhanced self-renewal capacity of hematopoietic stem cells (HSCs) in p18^-/- mice compared to wild-type (WT) mice. Given the critical role of bone marrow niche cells-particularly mesenchymal stromal cells (MSCs)-in hematopoiesis, this study investigated the functional alterations of p18^-/- MSCs and their impact on hematopoietic support.

Methods: Bone marrow derived MSCs were isolated from p18^-/- and WT mice. Their proliferation and differentiation capacities were assessed, followed by evaluation of hematopoietic support using cobblestone area-forming cell assay and long-term culture-initiating cell assay. RNA sequencing was performed to analyze the transcriptional profile of p18^-/- MSCs, with a focus on differentially expressed genes (DEGs). Key pathways associated with hematopoietic support were identified using Ingenuity Pathway Analysis. A candidate protein was quantified by ELISA, and its functional role in hematopoietic support was validated via a modified coculture system.

Results: p18^-/- MSCs displayed an increased proliferation rate, preferential differentiation toward osteogenesis over adipogenesis, and enhanced hematopoietic support. RNA sequencing analysis identified 137 DEGs, with secreted phosphoprotein 1 (Spp1, encoding osteopontin, Opn) being significantly upregulated in p18^-/- MSCs. Elevated Opn levels were confirmed in both bone marrow and MSC-conditioned media from p18^-/- mice. Functional validation further demonstrated that Opn enhanced the hematopoietic supportive capacity of MSCs in vitro.

Conclusions: p18 deficiency promotes osteogenic differentiation and enhances the hematopoietic supportive function of MSCs, likely mediated by Opn upregulation. These findings suggest a potential therapeutic strategy for improving bone regeneration and HSC expansion.