Neuroprotective and Neuroregenerative Effect of Secreted Factors from Human Adipose Stem Cells in a Rat Stroke Model

Abstract

Stem cell therapy is an emerging paradigm of stroke treatment. Recent evidence shows that stem cells exert their therapeutic effects through the secretion of immune modulatory, neurotrophic factors. Although potential neuroprotective and neuroregenerative mediators of stem cells have been previously suggested in both in vitro and in vivo studies, no study has compared their therapeutic effects with that of a whole stem cell secretome in an in vivo stroke model. Therefore, the objective of this study is to assess the neuroprotective and neuroregenerative effect of selected recombinant factors (RFs), detected in human adipose stem cell (hASC)-conditioned medium (CM), in a rat ischemic stroke model. The CM was prepared by culturing human adipose stem cells for 72h in serum-free Dulbeccos modified Eagle medium (DMEM). Multi-protein analysis using multiplex bead array and ELISA were used to detect secreted factors in the CM. Ischemic stroke was induced in Sprague-Dawley rats using 2 h transient middle cerebral artery occlusion (MCAO). In experiment 1, the vehicle (DMEM), concentrated CM, and selected RFs mixed with DMEM were administered intracerebroventricularly to each group (n = 14, 15, and 16, respectively) at 1 hr after reperfusion. Rats were sacrificed 24 h after MCAO. In experiment 2, the appropriate treatment was injected intraventricularly 24 h after reperfusion (n = 5, 4, and 4, respectively). All animals received daily single intraperitoneal injections of bromodeoxyuridine (BrdU, 50 mg/kg) from day 1 to day 3. Then, all animals exercised on a rat treadmill during 2 weeks and were sacrificed 17 day after reperfusion. IL-6, VEGF, HGF, and BDNF were detected in hASC-CM. In the experiment 1, the CM and RF groups both showed significantly better sensorimotor neurological test scores than the control group at 24 h post-MCAO. The infarct volume was significantly lower in both the CM and RF groups than in the control group. The number of TUNEL-positive apoptotic cells were reduced, whereas HSP70 expression was enhanced in the peri-infarct area in both the CM and RF groups. Moreover, hASC-CM and RFs reduced IκB phosphorylation and influenced bcl-2 and bax protein expression. In experiment 2, the RF group showed significantly better recovery of sensorimotor deficits at day 10, and increased neuronal proliferation in the perilesional area at day 17 after MCAO. Our results suggest that RFs, selected from hASC-CM, may exert an immediate neuroprotective effect in an ischemic stroke rat model that is comparable to those effect of full hASC-CM. The therapeutic effects of the RFs may be mediated by an anti-inflammatory mechanism and cell apoptosis inhibition. In addition, the selected RFs may also exert a neuroregenerative effect when administered after acute phase of stroke . Hence, treatment with RFs can be considered a feasible substitute for stem cell therapy after stroke.