we sought evidence that the functional recovery in animals with MSC-Ngn1 cells was due, at least in part, to neuronal differentiation of the transplanted cells. Evaluation of neuronal differentiation of grafted cells in vivo was performed. We identified MSC-Ngn1 cells were identified with anti-human mitochondrial antibody at 25days after transplantation. MSC-Ngn1 cells could be found in both cortical core and IBZ, where they differentiated into NeuN+ cells.
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Fig.20.Trans differentiation of MSCs/Ngn1 after intra-arterial administration.
Confocal images of the transplanted cells at 4 weeks after ischemia. MSC-Ngn1 cells were identified by the anti-NeuN antibody and human mitochondrial antibody. Arrowheads denote the donor-derived cells colocalized with cell type specific antigens in enlarged orthographic images.
61 neural induction of MSCs for better treatment of neurological dysfunctions.
We analyzed motor functions with Rotarod and adhesive removal test (Figure.1) from 1 day after ischemic stroke. Compared with the saline and MSC group, MSC-Ngn1 group showed significant recovery at day 7, which continued until day 28 after stroke in the both behavior tests. The infarct volumes of the ischemic animals were measured by MRI over the 4-week period monitoring the physical integrity of the brains. Animals grafted with MSC and MSC-Ngn1 cells showed higher recoveries than controls over the 4weeks. MSC cells reduced the infarct to a lesser degree compared with MSC-Ngn1 cells. These data indicated that the animals with MSC-Ngn1 cells exhibited the highest functional recovery among the tested animals.
From 4days after IA administration, MSC-Ngn1 group showed significant functional recovery comparing the other groups. That is not because of neuronal induction but the paracrine effects from the cells. We previously confirmed that MSC and MSC/Ngn1 have similar paracrine effects in the ischemic stroke. To check whether the MSC and MSC/Ngn1 differently distribute in the ischemic brain or not, we traced SPIO-labeled MSC and MSC-Ngn1 cells in the ischemic brain after intra-arterial administration (Figure 2). The cells could be detected as soon as 30min after IA injection scattering discretely throughout the ipsilateral side of the brain. While MSCs and MSC-ngn1 were shown similar amount and distribution pattern in the ipsilateral side until 4h after injection, a few MSC were seen from 1day comparing with the patterns of MSC-ngn1 cells. These data indicate that MSC-ngn1 were remained stable in the ischemic brain until 25 days after administration compared with MSCs. We also check the localization
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the cells labeled GFP at 1day and 4day after IA injection. Most of the transplanted cells were localized in the blood vessels at 1day and some cells were located near the blood vessels at 4days after IA injection. The SPIO-labeled cells could be detected through PB-staining and the signals were merged with hMT and ED1 indicating that some cells survived exhibiting human specific marker and the others were already died by macrophages at 25day after IA injections. These patterns were detected both MSCs and MSC-Ngn1 cells.
To compare the ability of cell migration under ischemic condition between MSC and Ngn1, we performed the migration assay using transwell chamber. MSC or MSC-Ngn1 cells was seeded in the upper Transwell chamber, while ischemic extract was added to the lower chamber. MSCs exhibited barely migration to the lower chamber, while MSC-Ngn1 showed high migratory activity to the ischemic extract. Verifying the differences of cell migration mechanism between MSC and MSC-Ngn1 after IA administration, we investigated the expression of chemoattractant receptors which involved in transmigration from blood vessels to ischemic brain, including CCR1, CCR2, CXCR4, CD44, MMP2, VLA-4 and c-Met in vitro. All of gene expressions in MSC/Ngn1 was higher than in MSCs. Especially CCR1, CCR2 and CXCR4 considering chemoattractant receptors were highly expressed in MSC/Ngn1. These data suggest that the better migration ability of MSC/Ngn1 in the ischemic brain results from the higher expressions level of receptors concerning cell adhesion, chemoattractant and transmigration.
It is known that MSCs produce bioactive cytokines that attenuate immune responds after ischemic injury and protect delayed cell death in the detrimental environment of damaged tissues. Though the MSC and MSC/Ngn1 exert similar paracrine effect in the ischemic brain, they differently remain in the ipsilateral side of the brain after IA administration. So we hypothesize that different amount of remaining cells cause different paracrine effect after IA transplantation. We evaluated the differences in inflammatory response 4 days after administrations. Active microglia, monocytes, and macrophages could be recognized by ED1 indicating ongoing inflammation.
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Comparing the animals with saline and MSC, the most numbers of ED1+ cells were reduced in the animals with MSC-Ngn1 cells. To confirm the anti-apoptotic effect in the ipsilateral regions, we performed TUNEL assays on the ischemic rat brains. The enormous apoptosis was shown in the control animals 1 week after injury. Intra-arterial injection of MSC and MSC-Ngn1 cells reduced the number of TUNEL+ cells considerably in the ischemic brain. Notably the higher reduction of TUNEL+ cells was shown in the animals with MSC-Ngn1 than MSC. These data indicate that MSCs and MSC-Ngn1 have paracrine effect on the ischemic brain 4 days after intra-arterial administration.
Ischemic brain at the level of the striatum were stained with anti-NeuN antibody to visualize the neuronal loss 4 weeks after the ischemic injury. In the saline group, most of NeuN+ cells in the cortex and striatum were lost. Some NeuN+ cells in the ischemic striatum were found in the MSC group indicating degeneration of striatum was less severe. Loss of NeuN+ cells was intensely reduced by transplantation of MSC-Ngn1 cells comparing the other groups. This data indicated that the higher recovery of behavioral impairment detected in the MSC-Ngn1 group is related to the higher tissue integrity of the host brain.
we sought evidence that the functional recovery in animals with MSC-Ngn1 cells was due, at least in part, to neuronal differentiation of the transplanted cells. Evaluation of neuronal differentiation of grafted cells in vivo was performed. We identified MSC-Ngn1 cells were identified with anti-human mitochondrial antibody at 25days after transplantation. MSC-Ngn1 cells could be found in both cortical core and IBZ, where they differentiated into NeuN+ cells.
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V. CONCLUSION
This study in an acute ischemic stroke model suggests that MSCs/Ngn1 stem cells have promising therapeutic effectiveness. IA administration would be a feasible grafting modality compared to IC administration, based on the behavior test results and MRI analysis.
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70 일에ratarod test와 adhesive removal test를포함한운동기능평가를시행하였다. Amimals는 3 개의하위그룹으로나뉘어졌습니다. 각군에는 1 × 106 개의 MSC-Ngn1 세포, 1 × 106 개 의 MSC-lacZ세포및정상생리식염수가각각투여되었다. 주입된줄기세포의분포를그룹간 비교하였다. MSC-Ngn1을주사한랫트는 MSC-LacZ및대조군과비교하여운동장애개선의