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. 2015 Nov 4:6:211.
doi: 10.1186/s13287-015-0202-2.

Adult bone marrow mesenchymal and neural crest stem cells are chemoattractive and accelerate motor recovery in a mouse model of spinal cord injury

Virginie Neirinckx  1 , Gulistan Agirman  2 , Cécile Coste  3 , Alice Marquet  4 , Valérie Dion  5 , Bernard Rogister  6   7   8 , Rachelle Franzen  9 , Sabine Wislet  10
Affiliations

Adult bone marrow mesenchymal and neural crest stem cells are chemoattractive and accelerate motor recovery in a mouse model of spinal cord injury

Virginie Neirinckx et al. Stem Cell Res Ther. .

Erratum in

Abstract

Introduction: Stem cells from adult tissues were considered for a long time as promising tools for regenerative therapy of neurological diseases, including spinal cord injuries (SCI). Indeed, mesenchymal (MSCs) and neural crest stem cells (NCSCs) together constitute the bone marrow stromal stem cells (BMSCs) that were used as therapeutic options in various models of experimental SCI. However, as clinical approaches remained disappointing, we thought that reducing BMSC heterogeneity should be a potential way to improve treatment efficiency and reproducibility.

Methods: We investigated the impact of pure populations of MSCs and NCSCs isolated from adult bone marrow in a mouse model of spinal cord injury. We then analyzed the secretome of both MSCs and NCSCs, and its effect on macrophage migration in vitro.

Results: We first observed that both cell types induced motor recovery in mice, and modified the inflammatory reaction in the lesion site. We also demonstrated that NCSCs but especially MSCs were able to secrete chemokines and attract macrophages in vitro. Finally, it appears that MSC injection in the spinal cord enhance early inflammatory events in the blood and spinal cord of SCI mice.

Conclusions: Altogether, our results suggest that both cell types have beneficial effects in experimental SCI, and that further investigation should be dedicated to the regulation of the inflammatory reaction following SCI, in the context of stem cell-based therapy but also in the early-phase clinical management of SCI patients.

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Figures

Fig. 1
Fig. 1
Characterization of MSCmix and NCSCmix isolated from the bone marrow of adult Wnt1-CRE/R26R-LacZ mice. After recombination, NCSCs from Wnt1-CRE/R26R-LacZ mice express LacZ gene. MSCs did not undergo Cre/Lox recombination and conserved the PGK-Neo cassette (a). MSCmix are adherent fibroblast-like cells, do not express β-galactosidase (b) or Sox2 c (red), slightly express Nestin (c) (green), p75NTR (d) (red), and Sca-1 (d) (green). NCSCmix have a similar morphology, express β-galactosidase (e), Nestin (f) (green), Sox2 (f) (red) and p75NTR (g) red), but not Sca-1(g) (green). Scale bar = 20 μm. MSC mesenchymal stem cell, NCSC neural crest stem cell
Fig. 2
Fig. 2
MSCmix and NCSCmix intraspinal cell graft accelerates motor recovery in mice with moderate SCI. MSC-grafted mice and NCSC-grafted mice recovered hindlimb motility faster than PBS-injected mice, as reflected by the Basso Mouse Scale scoring (a) (n = 6 to 7, repeated measures ANOVA and HSD post-test, *p < 0.05; **p < 0.01; ***p < 0.001). Indeed, it appears that MSC- and NCSC-grafted mice reach score 4 (plantar stepping, b) and score 6 (coordination, c) in a significantly reduced time interval (n = 6 to 7, one-way ANOVA and HSD post-test, **p < 0.01). MSC mesenchymal stem cell, NCSC neural crest stem cell, SCI spinal cord injury, PBS phosphate-buffered saline, ANOVA analysis of variance, HSD honestly significant difference
Fig. 3
Fig. 3
MSCmix and NCSCmix intraspinal cell graft influences lesion environment. After 3D reconstruction of spinal cord sections (a) stained with Luxol Fast Blue-Neutral Red (b), we evaluated the lesion volume in each group, which tends to be reduced in NCSC- (black) and MSC-grafted mice (dark grey) compared to controls (light grey) (b). We translated the evaluated lesion area on the next adjacent section to quantify the expression of GFAP, Iba1 and laminin. The GFAP-immunoreactive (ir) (c) and laminin-ir area (e) did not differ between the three groups, but the Iba1-ir area was a bit increased in MSC-grafted spinal cord sections (d). We also observed the presence of arginase 1-ir cells, not in control mice (f) but only in NCSC- (g) and MSC-grafted mice (h). The little rounded cells (h’) were more abundant in MSC- than in NCSC-grafted mice (i), and were recovered close to laminin-ir blood vessels (j), (k). (n = 6 to 7, one-way ANOVA and HSD post-test, *p < 0.05; ***p < 0.01). Scale bar = 500 μm (a to e), 200 μm (f to h), 20 μm (double, h’, j, k). MSC mesenchymal stem cell, NCSC neural crest stem cell, GFAP glial fibrillary acidic protein, ANOVA analysis of variance, HSD honestly significant difference
Fig. 4
Fig. 4
Qualitative and quantitative analysis of MSCmix- and NCSCmix-secretome. NCSCmix- and MSCmix-conditioned media (CM) are prepared by culturing MSCmix and NCSCmix in serum-free DMEM for 24 h (with or without stimulation by 1 μg/mL LPS: LPSMSC-CM and LPSNCSC-CM) and analyzed by Mouse Cytokine Array (a). Qualitative analysis of arrays revealed the presence of different chemokines and cytokines, especially present in MSC-secretome (b), and their concentration in each CM sample was assessed by ELISA assays. We observed an increased concentration of G-CSF (c), CXCL1 (d), CCL5 (e), IL-6 (f) in LPSMSC-CM, whereas CXCL12 was present in both MSC-CM and LPSMSC-CM (g). CXCL2 was present in very low concentrations in LPSMSC-CM and LPSNCSC-CM (h). CCL2 was more concentrated in NCSC-CM than in MSC-CM, but increased in both groups when prestimulated with LPS (i). TIMP-1 was equally secreted in all conditions (j), while M-CSF (k) and CXCL10 (l) were present in variable concentrations. (n = 5 to 7, one-way ANOVA and HSD post-test, *p < 0.05, **p < 0.01, ***p < 0.001). For values, see Additional file 1: Table S1. MSC mesenchymal stem cell, NCSC neural crest stem cell, DMEM Dulbecco’s modified Eagle’s medium, LPS lipopolysaccharide, ELISA enzyme-linked immunosorbent assay, G-CSF granulocyte colony-stimulating factor, CXCL C-X-C motif ligand, CCL C-C motif ligand, IL-6 interleukin-6, TIMP1, tissue inhibitor of metalloproteinases 1, ANOVA analysis of variance, HSD honestly significant difference
Fig. 5
Fig. 5
Effects of MSC- and NCSC-secretome on RAW264.7 macrophages migration and metabolic activity in vitro. We evaluated the chemoattractive properties of MSC and NCSC secretome by placing RAW264.7 macrophages on the top of a 5 μm-filter, with MSC-CM, LPSMSC-CM, NCSC-CM or LPSNCSC-CM in the bottom chamber (a). After 20 h, migration rate was assessed by the evaluation of the filter area occupied by macrophages. Passive migration was observed in response to serum-free DMEM (b, h), but drastically increased in response to LPS (c, h). MSC-CM slightly increased macrophage migration (d, h), but LPSMSC-CM was more efficient (e, h). NCSC-CM did not significantly induce macrophage migration (f, h), while LPSNCSC-CM also increased it (g, h). MTS metabolic assay also showed that LPSMSC-CM and LPSNCSC-CM increased the metabolic activity of RAW264.7 cells i (n = 3, one-way ANOVA and HSD post-test, **p < 0.01, ***p < 0.001). Scale bar = 50 μm. MSC mesenchymal stem cell, NCSC neural crest stem cell, CM conditioned medium, LPS lipopolysaccharide, DMEM Dulbecco’s modified Eagle’s medium, ANOVA analysis of variance, HSD honestly significant difference
Fig. 6
Fig. 6
Early immune cell recruitment is slightly increased in the blood and spinal cord of MSC-grafted mice. We collected spinal cords and plasma of mice, 24 h (white bars) and seven days (dark bars) post-injury (a). We observed that the concentration of G-CSF in the plasma of MSC-grafted mice was slightly increased after 4 h compared to other groups (b), and the concentration of sICAM-1 as well (c), while both markers returned to low levels after seven days. Additionally, the expression of Gr-1 (reflecting the presence of granulocytes) was slightly increased in MSC-grafted spinal cords at 24 h, and returned to low levels after seven days (d) while the expression of CD11b increased at this delay (e). Interestingly, the concentrations of CCL5 and CXCL1 were also slightly boosted after 24 h in MSC-grafted spinal cords (f, h), while CCL2 tended to increase in both MSC- and NCSC-grafted groups (g). MSC mesenchymal stem cell, G-CSF granulocyte colony-stimulating factor, sICAM1 soluble intercellular adhesion molecule, CCL C-C motif ligand, CXCL C-X-C motif ligand, NCSC neural crest stem cell
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