Bone marrow stromal cells promoting corticospinal axon growth through the release of humoral factors in organotypic cocultures in neonatal rats

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The transplantation of bone marrow stromal cells (BMSCs) is considered to be an alternative treatment to promote central nervous system regeneration, but the precise mechanisms of this regeneration after transplantation of BMSCs have not been clarified. In the present study, the authors assessed the effects of BMSC transplantation on corticospinal axon growth quantitatively, and they analyzed the mechanism of central nervous system regeneration in the injured and BMSC-treated spinal cord using the organotypic coculture system.


Bone marrow stromal cells derived from green fluorescent protein–expressing transgenic Sprague–Dawley rats were transplanted to the organotypic coculture system in which brain cortex and spinal cord specimens obtained in neonatal Sprague–Dawley rats were used. The axon growth from the cortex to the spinal cord was assessed quantitatively, using anterograde tracing with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate. To identify the differentiation of transplanted BMSCs, immunohistochemical examinations were performed. In addition, BMSCs were analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA expression of the growth factors.

The transplantation of BMSCs beneath the membrane, where the transplanted cells did not come into direct contact with the cultured tissue, promoted corticospinal axon growth to the same extent as transplantation of BMSCs on the tissues. The RT-PCR showed that the transplanted BMSCs expressed the mRNA of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF).


These findings strongly suggest that humoral factors expressed by BMSCs, including BDNF and VEGF, participate in regeneration of the central nervous system after transplantation of these cells.

Abbreviations used in this paper:BDNF = brain-derived neurotrophic factor; BMSC = bone marrow stromal cell; BMSC-UM = BMSC–under the membrane; DiI = 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; GalC = galactocerebroside; GFAP = glial fibrillary acidic protein; GFP = green fluorescent protein; MAP2 = microtubule-associated protein–2; NPC = neural progenitor cell; NT-3 = neurotrophin 3; PBS = phophate-buffered saline; RT-PCR = reverse transcriptase polymerase chain reaction; VEGF = vascular endothelial growth factor.

Article Information

Address reprint requests to: Naosuke Kamei, M.D., Ph.D., Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, 1–2–3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, 734–8551, Japan. email:

© AANS, except where prohibited by US copyright law.



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    Photomicrographic characterization of the cultured BMSCs. A: Differentiation of BMSCs into osteoblasts on incubation with osteogenic induction medium; calcium in the cultured differentiated BMSCs was detected by staining with alizarin red. B: Differentiation of BMSCs into adipocytes on incubation with adipogenic induction medium; fat droplets were detected in differentiated BMSCs on staining with Oil Red-O solution. C: The micromass that had been incubated in chondrogenic induction medium for 21 days showed proteoglycanrich matrix on staining with toluidine blue. D: Type II collagen (DAB) was detectable in the micromass of BMSCs. Bars = 100 μm.

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    A: Photomicrograph of a cortex–spinal cord organotypic coculture. B–E: The cocultures in the BMSC group were observed under a fluorescence microscope at 1 (B), 3 (C), 7 (D), and 13 (E) days after transplantation. A large number of GFP-positive cells were observed at the margin of the coculture tissues 1 day after transplantation, but the number of GFP-positive cells gradually decreased with time. F: The cocultures in the BMSCUM group were also observed under a fluorescence microscope at 1 day after transplantation. There were no GFP-positive cells around the coculture tissue. Arrows indicate the junction between the brain cortex and spinal cord. d = Day. GFP. Bars = 1000 μm (A) and 200 μm (B–F).

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    Immunostaining analyses. Cocultures treated with BMSCs were immunostained for nestin, MAP2, GalC, and GFAP 1, 3, 7, and 13 days after transplantation. The GFP-positive cells, indicating transplanted BMSCs, which were colabeled with nestin, were detected 3 and 7 days after transplantation; the GFP-positive cells that were colabeled with MAP2 and GalC were observed 3, 7, and 13 days after transplantation. However, there were no GFP-positive cells that were colabeled with GFAP at any time point. Green indicates the presence of GFP in all panels. A and B: Neural progenitor cells (red indicates nestin). C–E: Neurons (red indicates MAP2). F–H: Oligodendrocytes (red indicates GalC). Only one or two GFP-positive cells per culture labeled with nestin, MAP2, or GalC were detectable. Bars = 20 μm.

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    Axons projecting from the cortex to the spinal cord were revealed by DiI tracing. A: A limited number of axons extended into the substrate of the coculture in the control group. B: Axonal growth was enhanced after transplantation of BMSCs on the coculture. C: Axonal growth was also promoted after the transplantation of BMSCs-UM. Arrows indicate the junction between the brain cortex and spinal cord. Bars = 200 μm.

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    Bar graph providing quantitative assessment of axonal growth from the cortex to the spinal cord. The average number of axons in the BMSC and BMSC-UM groups was significantly greater than that in the control group at a distance of 500, 1000, 1500, and 2000 μm from the junction. There was no significant difference in axonal growth between the BMSC group and the BMSC-UM group. *p < 0.0001, **p < 0.0005, and ***p < 0.01.

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    Panel showing the mRNA levels of growth factors including BDNF, NT-3, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and VEGF, which were analyzed using RT-PCR. The mRNA expressions of BDNF, ciliary neurotrophic factor, and VEGF were detectable. GAPDH = glyceraldehyde-3-phosphate-dehydrogenase.


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