Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells

doi:10.21203/rs.3.rs-4690241/v1

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Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells

https://doi.org/10.21203/rs.3.rs-4690241/v1

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Background: The regenerative potential of mesenchymal stromal/stem cells (MSCs) has been extensively studied in clinical trials over the past decade. Despite the promising regenerative properties documented in preclinical studies, such as those for osteoarthritis (OA), the therapeutic translation of these results in patients has not been fully conclusive. One factor contributing to this therapeutic barrier could be the presence of senescent cells in the OA joint.

Methods: This study explores a novel approach to the treatment of OA by combining the therapeutic potential of adipose tissue-derived MSCs (AD-MSCs) with Rapamycin, a clinically approved immunosuppressive drug with anti-senescent properties. We first investigated the effects of Rapamycin on senescence and fibrosis markers in freshly isolated OA chondrocytes by immunostaining. We next evaluated the in vitro differentiation capacities of AD-MSCs, their regulatory immune functions on activated immune cells and their regenerative effects on OA chondrocyte signature in presence of Rapamycin.

Results: Rapamycin was found to reduce in OA chondrocytes the senescence marker p15ink4b

and the fibrotic marker COL1A1 in a dose-dependent manner without affecting the expression of the master chondrogenic markers SOX9 and COL2. The drug enhanced the differentiation of AD-MSCs into chondrocytes and reduced their adipogenesis. In addition, Rapamycin improved the immunoregulatory functions of AD-MSCs by promoting the expression of immunosuppressive elements such as IDO1, PTGS2, and PDL-1/CD274, the latter at the cell surface. Finally, by comparative RNA sequencing analysis, we revealed that AD-MSCs in presence of Rapamycin exhibited an improved chondroprotective regenerative effects on cocultivated OA chondrocytes.

Conclusions: Our findings propose that the combination of Rapamycin and AD-MSCs enhances the therapeutic efficacy of these cells on senescence-driven degenerative diseases such as OA. Overall, our data also open avenue for the identification of some factors that could contribute to the enhanced anti-fibrotic and anti-inflammatory properties of AD-MSCs in response to Rapamycin.

mesenchymal stromal/stem cells

Rapamycin

regenerative medicine

osteoarthritis

SuppFigure1.pdf
Supplementary Figure 1: Rapamycin has no effect on AD-MSC self-renewal. Primary AD-MSCs from healthy donors at passage 1 were seeded at 100 cells/9.6cm² (~10 cells/cm²) in the presence (or absence) of different concentrations of Rapamycin. Media were not removed along the experiment. After 10 days culture, colony were fixed, stained with Giemsa and counted. 0.01% DMSO (vehicle) treatment was used as a control. Mean effect on n=3 different AD-MSC donors, each performed in triplicate. Data were plotted and analyzed using GraphPad Prism 10.2.3 by Two-way ANOVA. (p-value: * <0.05, ** < 0.01, ***<0.001).
SuppFigure2.pdf
Supplementary Figure 2: Rapamycin does not significantly affect the expression of adipogenic markers. AD-MSCs in adipogenic and control conditions were treated the first 2 days after the induction of differentiation with 10nM rapamycin or 0.01% DMSO as vehicle control. At day 10, cells were lysed and the expression of PPARG2, FABP4, LEPTIN and ADIPOG were analysed by RTqPCR. RPLP0 was used as housekeeping genes. All error bars represent SEM and statistical analysis was performed by 2-way ANOVA using GraphPad Prism 102.3 (p-value: *<0.05, **<0.01, ***<0.001).
SuppFigure3.pdf
Supplementary Figure 3: RNAseq analysis reveals that Rapamycin sensitizes AD-MSCs to IFN-g response. (A) REACTOME pathway of all genes significantly induced by IFNγ treatment (FDR ≤ 0.05). (B) REACTOME pathway of the n=81 genes commonly induced by IFN-γ alone and IFN-γ+RAPA treatments (from the Venn diagram in Figure 3B), FDR ≤ 0.05.
SuppFigure4.pdf
Supplementary Figure 4: Defining OA versus healthy cartilage signature from GSE114007. Raw counts table from GSE114007 were retrieved and reanalysed using EdgeR. Reanalysis led to a list of 1956 upregulated and 1878 downregulated genes at FDR ≤ 0.05. (A) The n=1956 upregulated genes in OA were submitted to KEGG disease databank and significantly affected pathways (q-value ≤ 0.05) were presented as a bubble chart. The entire list of dysregulated genes in OA was submitted to KEGG pathway (B) and REACTOME (C), presented as a dual-sided histograms showing the number of genes affected in each significantly affected pathway (FDR ≤ 0.05). (D) Dot plots showing the normalized expression (log10(normalized counts+1)) in the n=18 healthy and n=20 OA cartilages of chondrogenic (SOX9), osteogenic (ALPL, SPARC, POSTN) and fibrotic (COL1A1, COL3A1, COL5A1, ELN) markers. Statistical comparisons were performed using Mann-Withney test. (P-value: * <0.05, **<0.01, ***<0.001).

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Reviewers agreed at journal
17 Jul, 2024
Reviewers invited by journal
17 Jul, 2024
Editor assigned by journal
05 Jul, 2024
First submitted to journal
05 Jul, 2024

You are reading this latest preprint version

Rapamycin improves the therapeutic regenerative potentials of mesenchymal stromal/stem cells

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