Transplants of human mesenchymal stem cells improve functional recovery after spinal cord injury in the rat

Abstract

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2x10(6) in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso-Beattie-Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.

Fig. 1.

hMSCs in the primary culture (A, C) and after cultivation at fourth passage (B, D–G). Primary culture contained heterogeneous population of flat adherent (thick arrow) (A) and small hematopoetic CD34+ cells (thin arrow) (C). Note, that almost pure population of slightly elongated flat fibroblastoid hMSCs appeared after fourth passage (thick arrow) with few residual CD34+ cells (thin arrow) (B, D). hMSCs immunoassayed with BrdU antibody indicating good quality of in vitro prelabeling and outlasting proliferation activity even at fourth passage (E). The hMSCs showed specific nuclei staining with human NUMA antibody by means of fluorescence and light immunocytochemical analysis (F, G). Nomarski objective (A, B, G). Scale bars: 100 μm, A, B; 50 μm, C–G.

Fig. 2.

Locomotor recovery of hindlimb function after SCI and hMSCs transplantation at 7 days evaluated in controls (n=15) and transplants (n=15) during 28 survival period. There were no significant differences of BBB scores between the groups at 14 days after injury (P > 0.05). Transplants reached significantly (P < 0.05) greater locomotor recovery at 21 and 28 days after SCI when compared with control group. The arrowhead on the abscissa indicates the time of transplantation. Data are presented as means ± SEM. Asterisk (*) denotes statistical differences (P < 0.05).

Fig. 3.

Expression of NUMA antibody in the coronal section of thoracic spinal cord (Th8) transplanted with hMSC. The majority of NUMA cells occurred in the ventral (A), lateral (B) white matter tracts, or occupied newly formed cavities (C). Boxed areas are taken from schematic spinal cord transversal section diagram A^*. The percent of NUMA (E) positive cells were calculated from total Dapi counterstained cells (D) taken from identical fields of ventro-lateral white matter, and coloclized with NUMA positivity (F). Scale bars: 500 μm, A^*; 100 μm, A–C; 25 μm, D–F.

Fig. 4.

NeuN (A, B) and MAP2 (E, F) staining of coronal thoracic spinal cord section taken from transplanted rats. Note, BrdU prelabeled hMSCs (red) distributed in the ventral (A) and dorsal horn (B) did not reveal NeuN expression (green). NUMA immunoreactive transplants (green, D) colocalized with MAP2 positivity in the lamina VII (F). Dapi counterstained nuclei in the identical sections (C). Scale bars 50 μm, A–F.

Fig. 5.

Expression of glial markers identified by GFAP (A) and APC (C, D) immunoreactivity in transplants. NUMA expressing hMSCs (arrows pointing to red nuclei) did not colocalized with GFAP (green) positivity (A). Double labeling of the sections with BrdU (B, green) and APC antibody (C, red) revealed that some grafted cells become oligodendrocytes (D). Scale bars 50 μm, A–D.

Fig. 6.

Distribution of GAP-43 positivity in coronal sections of controls (A, B) and transplants (C, D). Note, increased GAP-43 immunorectivity outlining regrowing axons within damaged dorsal (D) and lateral (C) white matter tracts after transplantation, but only moderate GAP-43 positivity in control rats subjected to trauma (A, B). Scale bars 100 μm, A, D; 50 μm, B, C.