Motor Recovery after Transplantation of Bone Marrow Mesenchymal Stem Cells in Rat Models of Spinal Cord Injury

Abstract

Background: Neuronal tissue has a limited potential to self-renew or get repaired after damage. Cell therapies using stem cells are promising approaches for the treatment of central nervous system (CNS) injuries. However, the clinical use of embryonic stem cells is limited by ethical concerns and other scientific consequences. Bone marrow mesenchymal stromal cells (BM-MSC) could represent an alternative source of stem cells for replacement therapy. Indeed, many studies have demonstrated that MSCs can give rise to neuronal cells as well as many tissue-specific cell phenotypes.

Purpose: Motor recovery by transplantation of bone marrow MSCs in rat models of spinal cord injury (SCI).

Methods: Bone marrow was collected from the femur of albino Wistar rats. MSCs were separated using the Ficoll-Paque density gradient method and cultured in Dulbecco's Modified Eagle Medium supplemented with 20% fetal bovine serum. Cultured MSC was characterized by immunohistochemistry and flow cytometry and neuronal-induced cells were further characterized for neural markers. Cultured MSCs were transplanted into the experimentally injured spinal cord of Wistar rats. Control (injured, but without cell transplantation) and transplanted rats were followed up to 8 weeks, analyzed using the Basso, Beattie, Bresnahan (BBB) scale and electromyography (EMG) for behavioral and physiological status of the injured spinal cord. Finally, the tissue was evaluated histologically.

Results: Rat MSCs expressed positivity for a panel of MSC markers CD29, CD54, CD90, CD73, and CD105, and negativity for hematopoietic markers CD34, CD14, and CD45. In vitro neuronal transdifferentiated MSCs express positivity for β III tubulin, MAP2, NF, NeuN, Nav1.1, oligodendrocyte (O4), and negativity for glial fibrillary acid protein. All the treated groups show promising hind-limb motor recovery BBB score, except the control group. There was increased EMG amplitude in treated groups as compared to the control group. Green fluorescent protein (GFP)-labeled MSC survived and differentiated into neurons in the injured spinal cord, which is responsible for functional recovery.

Conclusion: Our results demonstrate that BM-MSC has the potential to repair the injured cord in rat models of SCI. Thus, BM-MSC appears to be a promising candidate for cell-based therapy in CNS injury.

Keywords: Basso, Beattie, Bresnahan score; Electromyography; Mesenchymal stem cells; Spinal cord injury; Transplantation.

Fig. 1

Immunohistochemical characterization of rat MSC. Rat MSCs express positivity for the panel of markers CD29, CD54, CD90, CD73, and CD105 and negativity for hematopoietic markers CD34, CD14, and CD45. (Scale bar = 20 μm for CD29, CD54, and CD73 and 10 μm for CD90, CD105, CD34, CD14, and CD45).

Fig. 2

Surface marker expression of rat MSCs. Flow cytometry analysis of the immunophenotypic surface profiles for CD29, CD54, CD90, CD73, CD105, CD34, CD14, and CD45 of cultured MSCs. Histograms represent the counts of cells incubated with the relevant antibody. The logarithm on the x-axis represents the intensity of the fluorescent signal and the number of cells on the y-axis. Second passage cultured MSCs were positive for the markers CD29, CD54, CD73, CD90, and CD105 but negative for CD34, CD14, and CD45.

Fig. 3

Characterization of rat MSCs for neuronal markers. Photomicrographs of in vitro rat MSC showed negative for mature neuronal markers (MAP2, neurofilament, NeuN). Scale bar = 20 μm.

Fig. 4

In vitro differentiation of rat MSCs into neural cells. Phase-contrast image of undifferentiated rat MSCs and differentiated rat MSCs after 12 days in neuronal induction showing neural phenotype. Photomicrographs demonstrate that rat MSCs in neural induction medium differentiate into neural cells expressed neuronal proteins (β III tubulin, microtubule associated protein-2, neurofilament, NeuN), oligodendrocyte (O4), and negative for astrocyte (GFAP). Scale bar = 20 μm.

Fig. 5

Characterization of transdifferentiated MSCs. Immunofluorescence analysis of neuronal-induced rat MSCs indicates co-localization of (yellow) β III tubulin, a marker for neurons with (red) Nav1.1 (voltage-gated sodium channel). Scale bar = 20 μm.

Fig. 6

Patch-clamp studies. a Raw tracing from an MSC showing absence of inward currents at −40 mV and presence of outward currents at higher depolarizing potentials. The absence of voltage-gated sodium currents at the prepulse of −40 mV (shown by arrow) and the appearance of a family of depolarization-induced outward currents at depolarizing voltages. b Neuronal-induced MSCs show outward rectifying K⁺ current, absence of inward current at −40 mV (shown by arrow), and further depolarization. There is no voltage-gated sodium current seen.

Fig. 7

Viability of MSCs. Propidium iodide-stained cells show 3%, indicating dead cells, and remaining 97% of live cells as compared to unstained cells (control).

Fig. 8

Hind-limb motor recovery-mean BBB score. All the transplanted groups progressed in BBB score, except control group as shown in (a). In comparison within the groups before and after transplant, significant functional recovery was evident after transplant (p < 0.05; b). In comparison, between the groups, 5- and 10-lakh groups showed significant improvement in motor function (p < 0.05), when compared to the control group. But all the other transplanted groups showed functional recovery but statistically did not show significances (c). L, lakhs.

Fig. 9

Transcranial electrical stimulation and hind-limb motor evoked potential studies. Motor cortex stimulation and their responses were recorded in hind-limb muscle at the end of 8th week post transplant/SCI. On stimulation, there is no amplitude seen in control (a), but in transplanted rats EMG amplitude is shown with the arrow (b). Amplitude varies based on regeneration in different groups. Statistical analysis showed significant p < 0.05 in 5-lakh groups compared with control, whereas other groups did not show statistical significances but showed a remarkable increase in mean amplitude (c). L, lakhs.

Fig. 10

Histology of MSC-transplanted rat spinal cord. Green fluorescent protein (GFP)-labeled rat MSC in injured rat spinal cord. a Phase contrast. b GFP cells around the injury epicenter. c Merged image of (a) and (b). Immunohistochemistry on longitudinal sections of the injured spinal cord of rats 2 weeks after transplantation. Confocal immunofluorescence analysis of cryo sections indicates co-localization (green) GFP-labeled cells (d) with (red) βIII tubulin (e). Merged image (yellow) indicates differentiated neurons (f, g). Transplanted-rat MSC differentiates into neurons in vivo after SCI.