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. 2015:2015:945846.
doi: 10.1155/2015/945846. Epub 2015 Mar 26.

Skin-derived precursor cells promote angiogenesis and stimulate proliferation of endogenous neural stem cells after cerebral infarction

Duo Mao  1 , Xinpeng Yao  1 , Guowei Feng  2 , Xiaoqing Yang  3 , Lina Mao  1 , Xiaomin Wang  1 , Tingyu Ke  3 , Yongzhe Che  4 , Deling Kong  1
Affiliations

Skin-derived precursor cells promote angiogenesis and stimulate proliferation of endogenous neural stem cells after cerebral infarction

Duo Mao et al. Biomed Res Int. 2015.

Abstract

Stroke is one of the most common diseases that caused high mortality and has become burden to the health care systems. Stem cell transplantation has shown therapeutic effect in ameliorating ischemic damage after cerebral artery occlusion mainly due to their neurogenesis, immune regulation, or effects on the plasticity, proliferation, and survival of host cells. Recent studies demonstrated that skin-derived precursor cells (SKPs) could promote central nervous system regeneration in spinal cord injury model or the neonatal peripheral neuron. Here, we investigated the therapeutic potential of SKPs in a rat model of cerebral ischemia. SKPs were isolated, expanded, and transplanted into rat cortex and striatum after transient middle cerebral artery occlusion. Our results revealed that SKPs transplantation could improve the behavioral measures of neurological deficit. Moreover, immunohistology confirmed that SKPs could secrete basic FGF and VEGF in the ischemic region and further markedly increase the proliferation of endogenous nestin(+) and βIII-tubulin(+) neural stem cells. Furthermore, increased angiogenesis induced by SKPs was observed by vWF and α-SMA staining. These data suggest that SKPs induced endogenous neurogenesis and angiogenesis and protected neuron from hypoxic-ischemic environment. In conclusion, SKPs transplantation may be a promising approach in treatment of stroke.

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Figures

Figure 1
Figure 1
Phenotypic characterization and differentiation of SKPs. ((a)(I)) The appearance of SKPs by phase-contrast microscopy was neurospheres-like before passaging. ((a)(II), (a) (III)) SKPs that were adhered to a poly-d-lysine substratum overnight and then separately labeled with antibodies to nestin and fibronectin. ((a)(IV)–(VI)) Immunofluorescence colocalization analysis of SKPs showed coexpression of nestin (red) and P75 (green), and the nuclei were stained by DAPI. (b) Flow cytometric analysis of cell markers on nestin and α-SMA. Percentages indicate the fraction of cells that stained positive. (c) Differentiation of expanded SKPs into neural and mesodermal lineage cells in vitro. SKPs induced method was described in the “Section 2.” ((c)(I)–(III)) Immunostaining for SKPs, βIII-tubulin (red), the astrocyte marker GFAP (green), and smooth-muscle actin (α-SMA; red). ((c)(IV)) Adipogenesis was visualized by staining Oil red O. Scale bars, 10 μm.
Figure 2
Figure 2
Transplantation of SKPs ameliorates the behavioral impairments and reduces infarct volume in stroke model of rats. (a) Experimental study design. MCAO: middle cerebral artery occlusion; BrdU: bromodeoxyuridine. (b) Representation of the lateral ventricle wall that includes the stem cells injection site, neural progenitor cells (NPCs), the lateral ventricle (LV), the subventricular zone (SVZ), the ischemic boundary zone (IBZ), and the ischemic zone (IZ). ((c)(I), (c)(II)) Representative pictures of HE from animals treated with PBS or SKPs after MCAO. Scale bar, 1 mm. ((c)(III), (c)(IV)) Higher magnification showed that SKPs increased the number of cells with normal neuronal morphology and decreased the number of shrunken and misshapen cells in cresyl violet–stained sections. Scale bar, 10 μm. (d) Infarct size was measured on HE brain sections. Relative infarct size of PBS or SKPs-treated animals is presented as the mean ± S.D. The percentage of lesion tissue in the two groups (SKPs, Saline) at 14 days after occlusion. Two-way ANOVA with repeated measurements followed by one-way ANOVA and post hoc multiple comparison tests using Fisher's PLSD. (e) Behavioral performance in the neurological score (NSS) tests of PBS or SKPs injected animals from 1 to 14 days after ischemia. Statistically significant differences between the SKPs group with PBS group were determined by ANOVA, * P < 0.05.
Figure 3
Figure 3
Implanted SKPs secrete growth factors and differentiate into blood vessel in vivo. Immunostaining of DiI and growth factor bFGF, VEGF and blood vessel maker vWF, α-SMA in implantation site of IBZ. Brain sections were immunostained for ((a), (b)) bFGF, VEGF (green) or for ((c), (d)) vWF, α-SMA (green). Arrowhead shows DiI positive cells (red) that were colocalized with bFGF and VEGF (green). Scale bars, 10 μm.
Figure 4
Figure 4
SKPs increase SVZ and IBZ neurogenesis and neuronal progenitor migration to the ischemic lesion 14 days after MCAO. Immunostaining of neural stem cells markers and colocalization of BrdU in (a) SVZ and (b) IBZ of the ischemic rat brain. Brain sections were immunostained for both ((a)(I); (a)(III); (b)(I); (b)(III)) BrdU (green) and nestin (red) or for ((a)(II); (a)(IV); (b)(II); (b)(IV)) βIII-tubulin in SKPs group (top) and PBS (bottom). ((i), (ii)) Higher magnification of indicated by the white box. Arrowhead shows BrdU positive cells that were colocalized with nestin. Scale bars, 10 μm. (c) The pattern of implanted SKPs after cerebral brain ischemia. SVZ and IBZ were indicated by the black box, shadow area, and infracted zone. Quantitative analysis of (d) BrdU, (e) βIII-tubulin, and (f) nestin positive cells in the SVZ and IBZ (n = 5 for control and SKPs group). * P < 0.05, ∗∗ P < 0.01 versus control.
Figure 5
Figure 5
SKPs increase IZ and IBZ revascularization in MCAO model of rats. BrdU immunoreactive endothelial cells, α-SMA, and vWF vessels were detected in (a) IBZ and (b) IZ. Brain sections were immunostained for ((a)(I), (a)(III); (b)(I), (b)(III)) α-SMA or for ((a)(II), (a)(IV); (b)(II), (b)(IV)) both BrdU (green) and vWF (red) in SKPs group (top) and PBS (bottom). ((i), (ii), (iii), and (iv)) Higher magnification of indicated by the white box. Arrowhead shows BrdU positive cells that were colocalized with vWF. Scale bars, 10 μm. (c) The pattern of implanted SKPs after cerebral brain ischemia. SVZ and IZ were indicated by the black box, shadow area, and infracted zone. Quantitative data of number of (d) vWF or (e) α-SMA immunoreactive vessels and (f) BrdU immunoreactive endothelial cells. Injected SKPs (n = 5) significantly (P < 0.05) increased the number of endothelial cells and the density of vessels in the IZ and IBZ compared with group treated with PBS (n = 5).
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