Electrical stimulation affects the differentiation of transplanted regionally specific human spinal neural progenitor cells (sNPCs) after chronic spinal cord injury

Abstract

Background: There are currently no effective clinical therapies to ameliorate the loss of function that occurs after spinal cord injury. Electrical stimulation of the rat spinal cord through the rat tail has previously been described by our laboratory. We propose combinatorial treatment with human induced pluripotent stem cell-derived spinal neural progenitor cells (sNPCs) along with tail nerve electrical stimulation (TANES). The purpose of this study was to examine the influence of TANES on the differentiation of sNPCs with the hypothesis that the addition of TANES would affect incorporation of sNPCs into the injured spinal cord, which is our ultimate goal.

Methods: Chronically injured athymic nude rats were allocated to one of three treatment groups: injury only, sNPC only, or sNPC + TANES. Rats were sacrificed at 16 weeks post-transplantation, and tissue was processed and analyzed utilizing standard histological and tissue clearing techniques. Functional testing was performed. All quantitative data were presented as mean ± standard error of the mean. Statistics were conducted using GraphPad Prism.

Results: We found that sNPCs were multi-potent and retained the ability to differentiate into mainly neurons or oligodendrocytes after this transplantation paradigm. The addition of TANES resulted in more transplanted cells differentiating into oligodendrocytes compared with no TANES treatment, and more myelin was found. TANES not only promoted significantly higher numbers of sNPCs migrating away from the site of injection but also influenced long-distance axonal/dendritic projections especially in the rostral direction. Further, we observed localization of synaptophysin on SC121-positive cells, suggesting integration with host or surrounding neurons, and this finding was enhanced when TANES was applied. Also, rats that were transplanted with sNPCs in combination with TANES resulted in an increase in serotonergic fibers in the lumbar region. This suggests that TANES contributes to integration of sNPCs, as well as activity-dependent oligodendrocyte and myelin remodeling of the chronically injured spinal cord.

Conclusions: Together, the data suggest that the added electrical stimulation promoted cellular integration and influenced the fate of human induced pluripotent stem cell-derived sNPCs transplanted into the injured spinal cord.

Keywords: Cell therapy; Human induced pluripotent stem cells; Spinal cord injury; Spinal neuronal progenitor cells; Tail nerve electrical stimulation.

Fig. 1

Differentiation pattern of human iPSC-derived sNPCs at 21 days in vitro. Representative images of DAPI-labeled cells with neural markers: A GFAP, B APC, C MBP, D βIII-tubulin, and E NF200. Scale bar: 75 μm. The arrowheads indicate the highlighted cell (Boxed) in each image. F Percentage of differentiated cells

Fig. 2

Differentiation pattern of Human iPSC-derived sNPCs with or without TANES in chronically injured spinal cord of rats. Representative images depicting the SC121⁺ cells in the rostro-caudal direction to the lesion cavity of the spinal cord A sNPC only and B sNPC + TANES. Representative images of HNA or SC121-positive cells in green double labeled with GFAP, NF200, MBP, and synaptophysin (Syn) are represented in red in both C sNPC-only and D sNPCs + TANES groups. Scale bar: A, B 500 μm, C, D 75 μm. E Estimation of surviving cells utilizing cell density, F Quantification of the percentage of co-localization of human cells with specific markers (Ki67, Nestin, GFAP, NF200, APC, MBP, and Syn) in sNPC-only and sNPC + TANES groups. Data represent mean ± standard error of the mean; *p < 0.05, **p < 0.01; ns, non-significant

Fig. 3

Expression of synaptophysin (Syn) around transplanted sNPCs and host neurons. Representative image of the proximity of transplanted cells, host neurons and synaptophysin. A SC121, B NF200, C Syn, and D orthogonal view of the z stack image of SC121 (green), NF200 (red) and Synaptophysin (magenta) along with the orthogonal projection. Arrowheads indicate positively labeled cells. Scale bar: 75 μm. This finding suggests synapse formation between the transplanted cells and host cells

Fig. 4

Effect of TANES on survival and migration of transplanted sNPCs both rostral and caudal to the lesion site. Spinal cords sections demonstrating HNA (green) and DAPI (blue) 16 weeks post-transplantation rostral to the lesion site A, a sNPC only, B, b sNPC + TANES; caudal to the lesion cavity C, c sNPCs only, D, d sNPCs + TANES. Scale bar: A–D 250 μm; a–d 100 μm. E Quantification of migrated cells at 0.6 cm rostrally and caudally to the lesion cavity, F Percentage of cells with specific markers GFAP, MBP, and NF200 in sNPC-only or sNPC + TANES groups at 0.6 cm rostral and caudal to the lesion cavity. Data represent mean ± standard error of the mean; *p < 0.05; **p < 0.01; ns, non-significant

Fig. 5

Effect of TANES on the expression of serotonin-positive fibers (5HT) in the lumbar (L1–L3) region. Spinal cords were examined for the expression of serotonin-positive fiber (5HT, red) post treatment with either A injury only, B sNPC only, or C sNPC + TANES. a–c Higher-magnification images of the boxed areas. Scale bars: A–C 750 μm; a–c 200 μm. D Quantitative analysis revealed that expression of 5HT was significantly higher when rats received sNPCs and TANES. Data represents mean ± standard error. *p < 0.05; ns, non-significant

Fig. 6

Functional recovery analysis with BBB open field locomotor scores after combinatorial treatment with sNPC transplantation and TANES. There was a slight trend in the BBB scores from week 1 to week 16 when rats transplanted with sNPCs alone were compared with rats with injury alone. A much greater trend was observed when rats were stimulated with TANES, as compared with injury only