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. 2020 Sep 30;9(10):2218.
doi: 10.3390/cells9102218.

Short-Term Rapamycin Preconditioning Diminishes Therapeutic Efficacy of Human Adipose-Derived Stem Cells in a Murine Model of Multiple Sclerosis

Rachel M Wise  1   2 , Mark A A Harrison  1   2 , Brianne N Sullivan  1   2 , Sara Al-Ghadban  2   3 , Sarah J Aleman  1 , Amber T Vinluan  1 , Emily R Monaco  1 , Umberto M Donato  1 , India A Pursell  2 , Bruce A Bunnell  2   3   4
Affiliations

Short-Term Rapamycin Preconditioning Diminishes Therapeutic Efficacy of Human Adipose-Derived Stem Cells in a Murine Model of Multiple Sclerosis

Rachel M Wise et al. Cells. .

Abstract

Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cell populations in the spinal cord, and increased gene expression of interleukin-10 (IL-10), an anti-inflammatory cytokine. Conversely, EAE-Rapa-ASC mice showed no improvement in clinical disease scores, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning reduces the therapeutic efficacy of ASCs when applied to late-stage EAE.

Keywords: Rapamycin; adipose tissue-derived stem cells (ASCs); demyelination; experimental autoimmune encephalomyelitis (EAE); immunomodulation; inflammation; multiple sclerosis (MS).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rapa-ASCs do not reduce disease severity when administered at late-stage EAE. (A) Experimental design illustrating the timeline of EAE induction and schedule of clinical and behavioral assessments. (B) Severity of disease progression for each group over the course of 30 days, as determined by traditional clinical scoring system. (C) Balance and motor coordination evaluation with a fixed speed rotarod performance test. Reported as latency to fall. All data are presented as the mean ± SEM. Statistical analysis was performed using a mixed effects model of repeated measures analysis of variance (ANOVA) and Tukey’s post-hoc multiple comparisons test. Statistical differences between EAE and EAE-ASC are marked with * p < 0.05; ** p < 0.01. Statistical differences between EAE-ASC and EAE-Rapa-ASC are marked with # p < 0.05. Abbreviations: EAE, experimental autoimmune encephalomyelitis; Rapa, Rapamycin; MOG, myelin oligodendrocyte glycoprotein.
Figure 2
Figure 2
Rapa-ASCs result in reduced intact myelin when administered during late-stage EAE. Representative images of (A) hematoxylin and eosin-stained and (C) Luxol fast blue-stained lumbar spinal cord sections from vehicle-treated EAE (EAE), ASC-treated EAE (EAE-ASC) and Rapa-ASC-treated EAE (EAE-Rapa-ASC) mice. Quantitative comparison of (B) cellular infiltration and (D) myelin content in spinal cord sections between each group (n = 4). Quantitative data are represented as the number of positive pixels divided by the total pixels of the section. Statistical analysis was performed using a mixed effects model of repeated measures analysis of variance (ANOVA) and Tukey’s post-hoc multiple comparisons test. Statistical differences between the mean ± SEM of EAE and EAE-ASC are marked with ** p < 0.01; *** p < 0.001; and **** p<0.0001.
Figure 3
Figure 3
ASCs and Rapa-ASCs differentially regulate immune cell populations in the spleen and spinal cord 10 days post-treatment. (A) Cells isolated from spleens of vehicle-treated EAE (n = 5), ASC-treated EAE (n = 5), and Rapa-ASC-treated EAE (n = 6) mice were analyzed for canonical T cell markers with flow cytometry and represented as percentage of live cells. (B) Gene expression analysis of key T cell transcription factors in whole EAE spinal cords was performed using RT-qPCR. Data are presented as the mean ± SEM. Data are normalized to the housekeeping gene β-actin and represented as the relative fold change over vehicle-treated EAE controls. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and Tukey’s post-hoc multiple comparisons test. Statistical differences between groups are marked with * p < 0.05; ** p < 0.01. FOXP3, Forkhead Box P3; Tbet, T-box expressed in T cells; GATA3, GATA Binding Protein 3.
Figure 4
Figure 4
Gene expression analysis of T helper cell cytokines in the EAE spinal cord is altered by ASC treatments. Whole EAE spinal cords were analyzed using RT-qPCR for the T helper cell-derived cytokines TGF-β (A), IL-10 (B), and IL-4 (C). Data are presented as the mean ± SEM. Data are normalized to the housekeeping gene β-actin and represented as the relative fold change ± SEM over vehicle-treated EAE controls. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and Tukey’s post-hoc multiple comparisons test. Statistical differences between groups are marked with * p < 0.05; ** p < 0.01. TGF-β, transforming growth factor-beta; IL-10, interleukin-10; IL-4, interleukin-4.
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