Comparison of Mesenchymal Stromal Cells Isolated from Murine Adipose Tissue and Bone Marrow in the Treatment of Spinal Cord Injury

Abstract

The use of mesenchymal stromal cell (MSC) transplantation to repair the injured spinal cord has shown consistent benefits in preclinical models. However, the low survival rate of grafted MSC is one of the most important problems. In the injured spinal cord, transplanted cells are exposed to hypoxic conditions and exposed to nutritional deficiency caused by poor vascular supply. Also, the transplanted MSCs face cytotoxic stressors that cause cell death. The aim of this study was to compare adipose-derived MSCs (AD-MSCs) and bone marrow-derived MSCs (BM-MSCs) isolated from individual C57BL6/J mice in relation to: (i) cellular characteristics, (ii) tolerance to hypoxia, oxidative stress and serum-free conditions, and (iii) cellular survival rates after transplantation. AD-MSCs and BM-MSCs exhibited a similar cell surface marker profile, but expressed different levels of growth factors and cytokines. To research their relative stress tolerance, both types of stromal cells were incubated at 20.5% O₂ or 1.0% O₂ for 7 days. Results showed that AD-MSCs were more proliferative with greater culture viability under these hypoxic conditions than BM-MSCs. The MSCs were also incubated under H₂O₂-induced oxidative stress and in serum-free culture medium to induce stress. AD-MSCs were better able to tolerate these stress conditions than BM-MSCs; similarly when transplanted into the spinal cord injury region in vivo, AD-MSCs demonstrated a higher survival rate post transplantation Furthermore, this increased AD-MSC survival post transplantation was associated with preservation of axons and enhanced vascularization, as delineated by increases in anti-gamma isotype of protein kinase C and CD31 immunoreactivity, compared with the BM-MSC transplanted group. Hence, our results indicate that AD-MSCs are an attractive alternative to BM-MSCs for the treatment of severe spinal cord injury. However, it should be noted that the motor function was equally improved following moderate spinal cord injury in both groups, but with no significant improvement seen unfortunately following severe spinal cord injury in either group.

Keywords: Adipose-derived mesenchymal stromal cell (AD-MSC); bone marrow-derived mesenchymal stromal cell (BM-MSC); spinal cord injury (SCI); treatment.

Fig. 1.

Comparative analysis of mRNA expression of AD-MSCs and BM-MSCs using the QuantiGene Plex 2.0 Reagent System. The data showed that the expression of CCL2, CXCL12, PDGF-β, and VEGF-A in AD-MSCs were significantly higher than their expression in BM-MSCs. In contrast, BM-MSCs expressed significantly higher levels of BDNF than AD-MSCs. Data are shown as mean±SD. n=3 per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BDNF: brain-derived neurotrophic factor; BM-MSC: bone marrow-derived mesenchymal stromal cell; CXCL12: chemokine (C-X-C motif) ligand 12; PDGF-β: platelet-derived growth factor β; SD: standard deviation VEGF: vascular endothelial growth factor.

Fig. 2.

Assessment of stress tolerance of AD-MSCs and BM-MSCs under hypoxia and oxidative stress. (A) The proliferation rates of AD-MSCs and BM-MSCs under 20.5% O₂ or 1.0% O₂ culture conditions. At day 5, the number of AD-MSCs in 1.0% was significantly greater than AD-MSCs in 20.5% O₂. In contrast, the BM-MSC proliferation rate in 1.0% O₂ was significantly decreased at day 7. Data are shown as mean±SD, n=3 per groups, *p<0.05. (B) Representative images of AD-MSCs and BM-MSCs following 24 hours incubation in H₂O₂ containing DMEM/10% FBS medium or serum-free medium, stained with calcein AM/EthD-1 (LIVE/DEAD). Cells were photographed using a fluorescence microscope at 100× magnification. Living cells (green) were stained with calcein AM, and dead cells (red) were stained with EthD-1. (C, D) The number of the living/dead cells incubated in H₂O₂ at different concentrations or in serum-free medium. There were significantly greater numbers of viable AD-MSCs than BM-MSCs at the higher concentrations (750 μM, 1000 μM) of H₂O₂ and in serum-free conditions. Data are shown as mean±SD. n=6 wells per groups, *p<0.05. (E) Cell viability was further assessed using the XTT assay to test for metabolic activity. As shown, metabolic activity in AD-MSCs exposed to high concentrations of H₂O₂ (500–1000 μM) and in serum-free medium was significantly greater than that of BM-MSCs under the same stress condition. Data are shown as mean±SD. n=6 wells per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BM-MSC: bone marrow-derived mesenchymal stromal cell; DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; XTT: 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide.

Fig. 3.

The survival of AD-MSCs and BM-MSCs after transplantation into the injured spinal cord. (A) Representative images are shown of midsagittal spinal cord sections in mice transplanted with GFP-labeled AD-MSCs and GFP-labeled BM-MSCs in the severe SCI model. High-magnification pictures were taken from respective boxed area showing GFP-MSCs with cell protrusions. Scale bars: 200 µm, 20 μm. (B) The midsagittal GFP-positive area was analyzed after transplantation. There was a significantly higher number of positive cells of GFP-labeled AD-MSCs than GFP-labeled BM-MSCs at 2, 3, and 4 weeks after transplantation. Data shown as means±SD. n=4 per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BM-MSC: bone marrow-derived mesenchymal stromal cell; GFP: green fluorescent protein; SCI: spinal cord injury; SD: standard deviation.

Fig. 4.

Akt and ERK 1/2 immunoreactivity in transplanted AD-MSCs and BM-MSCs. (A) Representative immunofluorescence images show the colocalization of Akt and ERK1/2 (red) GFP-positive MSC (green) with cell protrusions in the spinal cord epicenter on 7 days after transplantation. Scale bars: 20 µm. (B) The number of Akt or ERK1/2 and GFP-positive MSCs. The amount of GFP-AD-MSC colocalization with Akt or ERK1/2 was significantly increased compared with that of GFP-BM-MSCs. Data are shown as mean±SD. n=3 per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BM-MSC: bone marrow-derived mesenchymal stromal cell; GFP: green fluorescent protein; MSC: mesenchymal stromal cell.

Fig. 5.

The effects of AD-MSC and BM-MSC transplantation on corticospinal tracts and blood vessel formation in severe SCI. (A) Representative images are shown for each experimental group of the corticospinal tracts ± 500 μm from the injury epicenter at 4 weeks after transplantation. Scale bar: 50 μm. (B) Immunofluorescence positive area of PKCγ (red) merged with axons in YFP-mice at 2, 4, and 6 weeks after AD-MSC and BM-MSC transplantation versus controls (no cells). Data showed that the PKCγ-positive corticospinal tracts were significantly increased at 2, 4, and 6 weeks after transplantation in the AD-MSC transplanted group compared with the BM-MSC transplanted group and control group. (C) Representative images showing CD31 immunofluorescence staining (red) at 1 and 2 weeks after transplantation of AD-MSCs or BM-MSCs versus controls (no cells). CD31 positive blood vessels area and tube formation were increased in the AD-MSC transplanted group. Scale bar: 50 μm). (D) Immunofluorescence positive area of CD31 on 1 and 2 weeks after transplantation. Note the CD31 positive capillary density was significantly increased in AD-MSC transplanted group compared with the BM-MSC transplanted group and control group. Data are shown as mean±SD. n=5 per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BM-MSC: bone marrow-derived mesenchymal stromal cell; PKCγ: anti-gamma isotype of protein kinase C; SCI: spinal cord injury; SD: standard deviation.

Fig. 6.

The effects of AD-MSCs and BM-MSCs after transplantation on hind limb function. (A) BMS scores after MSC transplantation following mild contusion SCI (60 kdyne contusion). Both MSC treatment groups showed significantly better motor function than the control group. (B) BMS score after MSC transplantation following severe contusion SCI (80 kdyne contusion). There were no significant differences between each of the groups. Data are show as mean±SD. n=10 per groups, *p<0.05. AD-MSC: adipose-derived mesenchymal stromal cell; BM-MSC: bone marrow-derived mesenchymal stromal cell; BMS: Basso Mouse Scale; MSC: mesenchymal stromal cell; SCI: spinal cord injury; SD: standard deviation.