Stem cells from human exfoliated deciduous tooth-derived conditioned medium enhance recovery of focal cerebral ischemia in rats

Abstract

Regenerative therapy using stem cells is a promising approach for the treatment of stroke. Recently, we reported that dental pulp stem cells (DPSC) ameliorated ischemic tissue injury in the rat brain and accelerated functional recovery after middle cerebral artery occlusion (MCAO). In this study, we investigated the effects of stem cells from human exfoliated deciduous tooth (SHED)-derived conditioned medium (SHED-CM) on permanent MCAO (pMCAO). Adult male Sprague-Dawley rats were subjected to pMCAO. SHED-CM were then administered intranasally, and the motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined using immunochemical markers. The SHED-CM group had more positive signals than the Dulbecco's modified Eagle's medium group, with doublecortin (DCX), neurofilament H, neuronal nuclei, and rat endothelial cell antigen observed in the peri-infarct area. Migration of neuronal progenitor cells (NPC) with DCX from the subventricular zone to the peri-infarct area was observed on days 6 and 16, with migration on day 6 being the most prominent. In conclusion, SHED-CM promoted the migration and differentiation of endogenous NPC, induced vasculogenesis, and ameliorated ischemic brain injury after pMCAO as well as transplantation of DPSC.

FIG. 1.

(A) The peri-infarct area. Peri-infarct area (gray) and infarct core (black). (B) Anatomy around the olfactory bulb. 1: nasal bone. 2: forehead bone. 3: ethmoid bone. 4: olfactory nerve area. 5: olfactory bulb. 6: forehead bone. 7: cerebrum. 8: cerebellum. 9: incisor teeth. 10: incisive bone. 11: maxilla bone. 12: molar teeth. The arrow displays the passage of nasal administration.

FIG. 2.

Motor disability test following intranasal administration of stem cells from human exfoliated deciduous tooth (SHED)-derived conditioned medium (SHED-CM), bone marrow mesenchymal stem cell (BMMSC)-derived conditioned medium (BMMSC-CM), and Dulbecco's modified Eagle's medium (DMEM) on days 1, 3, 6, 9, 12, and 15. Data are expressed as means±SD of 3 determinations. *p<0.05, versus DMEM. ^#p<0.05 versus BMMSC-CM. Student's t-test.

FIG. 3.

Reduction in the infarct volume 16 days after the injection of SHED-CM, BMMSC-CM, and DMEM (A). Infarct area on day 16 after nasal injection (B:DMEM, C:BMMSC-CM, and D:SHED-CM). Data are expressed as means±SD of 3 determinations (A). *p<0.05, **p<0.01, versus DMEM, ^#p<0.05, versus BMMSC-CM. Student's t-test.

FIG. 4.

Doublecortin (DCX)-positive cells (A, B), neurofilament H (NF)-positive cells (C, D), neuronal nucleus (NeuN)-positive cells (E, F), and rat endothelial cell antigen (RECA1)-positive cells (G, H). SHED-CM group (A, C, E, G) on day 16. DMEM group (B, D, F, H) on day 16. Bar=50 μm (A–H). Cell nuclei were counterstained with DAPI. Statistical analyses of density of DCX (I), NeuN (J), and RECA1 (K) on day 16. *p<0.05, **p<0.001, Student's t-test. Each point is expressed as the mean±SD of 75 determinations. Color images available online at www.liebertpub.com/tea

FIG. 5.

Migration of neuronal progenitor cells (NPC) from the SVZ to the peri-infarct area on days 6 (A, B) and 16 (C, D). SHED-CM intranasal group (A, C). DMEM intranasal group (B, D). Bar=100 μm (A–D). IA=infarct area. Color images available online at www.liebertpub.com/tea