Human Adipose Stromal/Stem Cells from Obese Donors Show Reduced Efficacy in Halting Disease Progression in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

Abstract

Multiple sclerosis is an autoimmune disease that affects the white matter of the central nervous system and involves inflammation and demyelination. The recent advances in our understanding of adipose-derived stromal/stem cells (ASCs) and the utilization of these cells in clinical settings to treat diseases have made it essential to identify the most effective ASCs for therapy. Studies have not yet investigated the impact of obesity on the therapeutic efficacy of ASCs. Obesity is characterized by adipocyte hyperplasia and hypertrophy and can extend to metabolic and endocrine dysfunction. Investigating the impact obesity has on ASC biology will determine whether these cells are suitable for use in regenerative medicine. The therapeutic efficacy of ASCs isolated from lean subjects (body mass index [BMI] < 25; lnASCs) and obese subjects (BMI > 30; obASCs) were determined in murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Compared with the EAE disease-modifying effects of lnASCs, obASCs consistently failed to alleviate clinical symptoms or inhibit inflammation in the central nervous system. When activated, obASCs expressed higher mRNA levels of several pro-inflammatory cytokines compared with lnASCs. Additionally, conditioned media (CM) collected from the obASCs markedly enhanced the proliferation and differentiation of T cells; whereas, CM from lnASC did not. These results indicate that obesity reduces, or eliminates, the anti-inflammatory effects of human ASCs such that they may not be a suitable cell source for the treatment of autoimmune diseases. The data suggest that donor demographics may be particularly important when identifying suitable stem cells for treatment.

Keywords: Adipose stromal/stem cells; Adipose-derived stromal/stem cells; Experimental autoimmune encephalomyelitis; Immunomodulatory properties; Multiple sclerosis; Obesity; Therapeutic efficacy.

Figure 1

Lean ASCs (lnASCs), but not obese ASCs, delay disease onset and reduce disease severity. (A): EAE mice were treated with Hanks' balanced saline solution (HBSS) (n = 7), lnASCs (n = 8), or obASCs (n = 7) on 0 days post induction (DPI) as indicated by the arrow and clinical scores were collected daily for the duration of the study. (B): The number of EAE mice with each clinical score (0–4) is shown for each treatment group 30 DPI. (C): Track visualizations were generated for each mouse every 5 days using EthoVision XT7 and representative images of 5-minute intervals for each respective day are shown. (D): Parameters of motor function were assessed with Ethovision XT7. Bar ± SEM. *, p < 0.05, compared with the HBSS group. #, p < 0.05, compared between the lnASC and obASC groups.

Figure 2

lnASC, but not obASC, reduce inflammation, limit size of lesions, and improve myelin levels. EAE induced mice treated at the onset of the disease with Hanks' balanced saline solution (HBSS) (n = 7), lnASC (n = 8), or obASC (n = 7). Spinal cords were harvested on 30 DPI. (A): Representative histological slides stained with H&E or luxol fast blue. Arrows indicate lesions and arrowhead indicates perivascular infiltration. Solid lines indicate demyelinated area. Scale bar = 100 μm. (B): Quantitative analysis of histological stains demonstrating number of lesions, average area per lesion, and total area occupied by lesions. Bar ± SEM. *, p < 0.05; ***, p < 0.001, compared with the HBSS group. #, p < 0.05; ##, p < 0.01, compared between the lnASC and obASC groups. Abbreviations: HE, Hematoxylin and Eosin; LFB, Luxol Fast Blue.

Figure 3

ASC treatment reduces lymph node size and increases splenic weight. EAE mice were treated with Hanks' balanced saline solution (HBSS), lnASCs, or obASCs on 0 DPI (before disease onset) and organs were collected 30 DPI for assessment. (A): Lymph nodes and spleens were weighed. (B): Representative images of lymph nodes and spleens are shown. Scale bar = 200 μm. Bar ± SEM. *, p < 0.05, compared with the HBSS group. #, p < 0.05, compared between the lnASC and obASC groups.

Figure 4

obASCs enhance proliferation and differentiation of mouse T cells. T cells were isolated from EAE mice 30 DPI and exposed to water (vehicle control) or 20 μg/ml of myelin oligodendrocyte glycoprotein_35-55 (MOG_35-55). After overnight stimulation, T cells were exposed to conditioned media (CM) from lnASCs (lnASC CM) or obASCs (obASC CM). T cells exposed to serum free αMEM were used as a control. (A): After 48 hours, cells were incubated in Alamar blue to assess proliferative rate. Values are shown as relative fluorescence unit acquired at an excitation wavelength of 544 nm and emission wavelength of 584 nm. (B): T cells induced with MOG_35-55 and exposed to CM were assessed for cell surface marker expression. Data are shown relative to the total percentage of CD3⁺ cells. Values represent triplicates conducted for each of the three EAE mice (n = 3). Bar ± SEM. **, p < 0.01; ***, p < 0.001, compared with no CM group (control). ^#, p < 0.05; ^###, p < 0.001, compared between lnASCs CM and obASC CM groups.

Figure 5

mRNA expression of pro-inflammatory cytokines is higher in IFN-γ obASCs than lnASCs induction. lnASCs (n = 6 donors) and obASCs (n = 6 donors) were treated with vehicle control (water) or 5 ng/ml of IFN-γ. After 48 hours, cells were harvested and analyzed by qRT-PCR. Data are normalized to vehicle-treated lnASCs. Bar ± SEM. **, p < 0.01; ***, p < 0.001, between non-induced and induced cells. ^##, p < 0.01; ^###, p < 0.001, between induced lnASCs and induced obASCs. Abbreviations: IL-1, interleukin-1; IL-6, interleukin-6; IL-12, interleukin-12; PDGF-A, • • •; TNF-α, tumor necrosis factor-α; LIF, Leukemia Inhibitory Factor; ICAM-1, Intercellular Adhesion Molecule 1; G-CSF, Granulocyte-colony stimulating factor.

Figure 6

lnASCs, but not obASCs, reduce disease severity. (A): EAE mice were treated with Hanks' balanced saline solution (HBSS) (n = 8), lnASCs (n = 8), or obASCs (n = 7) on 15 days post induction (DPI) as indicated by the arrow and clinical scores were collected daily for the duration of the study. (B): The number of EAE mice with each clinical score (0–4) is shown for each treatment group 30 DPI. (C): Track visualizations were generated for each mouse every 5 days using EthoVision XT7 and representative images of 5-minute intervals for each respective day are shown. Bar ± SEM. *, p < 0.05, compared with the HBSS group.

Figure 7

obASC fail to reduce inflammation, limit size of lesions, and preserve myelin. EAE mice treated on DPI 15 with Hanks' balanced saline solution (HBSS) (n = 8), lnASC (n = 8), or obASC (n = 7). Spinal cords were harvested on 30 DPI. (A): Representative histological slides stained with H&E or luxol fast blue. Arrows indicate lesions and arrowhead indicates perivascular infiltration. Solid lines indicate demyelinated area. Scale bar = 100 μm. (B): Quantitative analysis of histological stains demonstrating number of lesions, average area per lesion, and total area occupied by lesions. Bar ± SEM. *, p < 0.05; ** p <0.01; ***, p < 0.001, compared with the HBSS group. Abbreviations: HE, Hematoxylin and Eosin; LFB, Luxol Fast Blue.