Human umbilical cord blood-derived mesenchymal stem cell therapy promotes functional recovery of contused rat spinal cord through enhancement of endogenous cell proliferation and oligogenesis

Sang In Park, Jung Yeon Lim, Chang Hyun Jeong, Seong Muk Kim, Jin Ae Jun, Sin-Soo Jeun, Won Il Oh, Sang In Park, Jung Yeon Lim, Chang Hyun Jeong, Seong Muk Kim, Jin Ae Jun, Sin-Soo Jeun, Won Il Oh

Abstract

Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.

Figures

Figure 1
Figure 1
BBB scores of rats with SCI before and after hUCB-MSCs transplantation at 7 days after SCI. (a) hUCB-MSC transplantation group displayed significantly improved scores compared with control at 6 weeks after transplantation. (b) Cavity volume between the hUCB-MSC and control groups at 6 weeks after transplantation. The values of the cavity volume of the hUCB-MSC group were lower than those of the control group. (c) and (d) HE-stained sections of transplantation group and control group, P < 0.05.
Figure 2
Figure 2
Quantitative analysis of BrdU-labeled cells in the ependymal and parenchymal regions. (a) Result of immunohistochemistry using anti-BrdU antibody. (b) Enlargement of the boxed region in (a), showing BrdU-labeled cells in the parenchymal region. (c) Average number of BrdU-labeled cells per white matter area from all five white matter areas in the parenchymal region. (d) Average number of BrdU-labeled cells per ependymal region in grey matter. At 14 days after transplantation, proliferation of endogenous cells was significantly increased from injury site to cell transplantation site in hUCB-MSCs-transplanted group compared with control group, *P < 0.05.
Figure 3
Figure 3
Endogenous neurogenesis induced by transplantation. Endogenous stem cells were assessed quantitatively by double staining of BrdU with nestin, GFAP, and NG2 at 1 and 2 weeks after transplantation in both the ependymal and parenchymal regions. (a)–(d) At 1 and 2 weeks following transplantation, BrdU/nestin-labeled cells as well as BrdU/GFAP-labeled astrocytes were present in ependyma. (e) and (h) BrdU-labeled NG2 cells were coexpressed at 1 and 2 weeks in the parenchyma. (i) and (j) The numbers of BrdU-labeled ependyma coexpressing GFAP/nestin were quantified at 1 and 2 weeks after transplantation. (k) The numbers of BrdU-labeled parenchyma coexpressing NG2 were quantified at 1 and 2 weeks after transplantation. *P < 0.05, scale bars = 10 μm in (a)–(d); 20 μm in (e)–(h).
Figure 4
Figure 4
Quantitative analysis of endogenous oligogenesis by hUCB-MSCs. At 2 weeks after cell transplantation, BrdU and cell-specific markers were observed up to the edge of the SCI region. (a) and (b) BrdU/CNPase-labeled cells were present. (c) and (d) BrdU/MBP-labeled cells. (e) and (f) BrdU/GFAP-labeled cells. (g) Quantity of BrdU/CNPase, MBP, and GFAP-labeled cells. *P < 0.05. Scale bars: 10 μm.
Figure 5
Figure 5
Protection of apoptosis by hUCB-MSCs as revealed by TUNEL assay in the injury site at 2 weeks after transplantation. (a)–(d) TUNEL staining (green) and staining with 4′,6-diamidino-2-phenylindole (blue) indicate undergoing apoptotic cell death. (e) Quantity of TUNEL positive cells. The number of TUNEL positive cells was significantly reduced in cell transplantation group than in control group. *P < 0.05, scale bars denote: 10 μm.

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