Transplantation of predifferentiated adipose-derived stromal cells for the treatment of spinal cord injury

Abstract

Adipose-derived stromal cells (ASCs) are an alternative source of stem cells for cell-based therapies of neurological disorders such as spinal cord injury (SCI). In the present study, we predifferentiated ASCs (pASCs) and compared their behavior with naïve ASCs in vitro and after transplantation into rats with a balloon-induced compression lesion. ASCs were predifferentiated into spheres before transplantation, then pASCs or ASCs were injected intraspinally 1 week after SCI. The cells' fate and the rats' functional outcome were assessed using behavioral, histological, and electrophysiological methods. Immunohistological analysis of pASCs in vitro revealed the expression of NCAM, NG2, S100, and p75. Quantitative RT-PCR at different intervals after neural induction showed the up-regulated expression of the glial markers NG2 and p75 and the neural precursor markers NCAM and Nestin. Patch clamp analysis of pASCs revealed three different types of membrane currents; however, none were fast activating Na(+) currents indicating a mature neuronal phenotype. Significant improvement in both the pASC and ASC transplanted groups was observed in the BBB motor test. In vivo, pASCs survived better than ASCs did and interacted closely with the host tissue, wrapping host axons and oligodendrocytes. Some transplanted cells were NG2- or CD31-positive, but no neuronal markers were detected. The predifferentiation of ASCs plays a beneficial role in SCI repair by promoting the protection of denuded axons; however, functional improvements were comparable in both the groups, indicating that repair was induced mainly through paracrine mechanisms.

Fig. 1

a Spheres were all positive for NCAM (red). Dissociated spheres plated on laminin-coated coverslips (b, c) and fixed after 6 days in neural medium (d). b S100 (green) NG2 (red); c staining for p75 (red), GFP (green); d NCAM (red), βIII-tubulin (green). e GFP⁺ ASCs (green) 8 weeks after transplantation into a SCI did not interact with host axons (staining for NF160; red); inset is at higher magnification and shows the lack of interaction between an ASC and the host tissue. f–h In contrast, GFP⁺ pASCs (green) formed hollow fibers and closely wrapped (white arrows) host axons (f, g; staining for NF160, red) as well as oligodendrocytes (h; staining for O1, red). Inset at higher magnification shows the close interaction of GFP⁺ cells with neurofilaments. i Some pASCs were positive for NG2 (i, white arrows) and formed guiding strands along the residual host tissue, attracting host NG2-positive progenitors (red) to the lesion site. j TEM image showing a GFP⁺ pASC (arrow) in close contact with a Schwann cell (S) and a myelinated axon (a). k GFP⁺ process from a pACS (arrow) in the vicinity of a Schwann cell (S) and an unmyelinated axon (a). l, m Both ASCs (L) as well as pASCs (M) were positive (arrows) for the endothelial marker CD31 (red). Cell nuclei are stained in blue (DAPI). Scale barsa–e, i—20 μm, f, g—50 μm, j—1 μm, k—2.5 μm, h, l, m—25 μm

Fig. 2

a–c Quantitative RT-PCR analysis of spheres and dissociated cells from the spheres maintained in neural medium, 48 h and 6 days after NI. The enhanced expression of glial markers and immature neural progenitor markers was found after exposure to differentiation medium. d BBB open-field locomotor test. Hind limb gait was first assessed 1 week after SCI, i.e., before transplantation (Tx), and evaluated weekly thereafter for 7 weeks. Both the ASC- (BBB = 7.0 ± 0.74; n = 11) and the pASC- (BBB = 7.2 ± 0.46; n = 12) treated groups displayed motor improvement compared with controls (BBB = 4.7 ± 0.34; n = 12). There was no significant difference between the transplanted groups