Volume 9, Issue 2, Pages 189-197 (February 2004) BDNF Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model  Kazuhiko Kurozumi, Kiminori Nakamura, Takashi Tamiya, Yutaka Kawano, Masayoshi Kobune, Sachie Hirai, Hiroaki Uchida, Katsunori Sasaki, Yoshinori Ito, Kazunori Kato, Osamu Honmou, Kiyohiro Houkin, Isao Date, Hirofumi Hamada  Molecular Therapy  Volume 9, Issue 2, Pages 189-197 (February 2004) DOI: 10.1016/j.ymthe.2003.10.012 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 1 BDNF production by cultured MSC. MSC transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at m.o.i. of 100, 300, 1000, and 3000 pu/cell secreted BDNF at a rate of 0.230 ± 0.110, 0.434 ± 0.122, 0.931 ± 0.101, and 1.860 ± 0.41 ng/105 cells/48 h, respectively. Nontransfected MSC also produced BDNF (0.0407 ± 0.0059 ng/105 cells/48 h). Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Assessment of brain ischemia-induced neurological deficits. (A) Deficits in limb placement were evaluated using the following scale: 0, severe neurological deficits; 16, no neurological deficits. One day after MCAO but prior to intracerebral MSC injection, there was no statistical difference in limb placement score among the four ischemic groups. Eight days after MCAO, rats that received MSC-BDNF achieved significantly higher limb placement scores compared to control DMEM rats (P = 0.0001) and rats that received fibroblasts (P = 0.003). Fifteen days after MCAO, the score of rats that received MSC-BDNF was similarly elevated compared to the DMEM group (P = 0.024). (B) Prior to MCAO, mean treadmill speeds were comparable between groups. Eight days after MCAO, rats in the MSC-BDNF group achieved significantly higher speeds compared to animals in the control DMEM- (P = 0.001) and fibroblast-treated (P = 0.017) groups. These differences were maintained on day 15, with speeds in the MSC-BDNF group being significantly higher than in the control DMEM (P = 0.002) and fibroblast groups (P = 0.023). Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 3 (A) T2-weighted (T2W) images taken 2, 7, and 14 days after MCAO in rats injected with DMEM, fibroblasts, MSC, or MSC-BDNF. Seven days after MCAO, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to rats that received DMEM (P = 0.002), fibroblasts (P = 0.015), or MSC (P = 0.028). After 14 days, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to those that received DMEM (P = 0.011). (B) Representative T2W images obtained 2 and 7 days after MCAO in rats injected with DMEM, MSC, or MSC-BDNF. A reduction in the volume of ischemic damage was detected in the MSC-BDNF group compared to the other groups on day 7. Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 4 In vivo levels of BDNF production. BDNF levels were significantly elevated in the ischemic hemisphere of MSC-BDNF-transplanted rats 7 days after MCAO compared to rats that received DMEM (P = 0.0002) or MSC (P = 0.0006). Levels of BDNF were also significantly increased in the ischemic hemisphere of MSC compared to DMEM-treated rats (P = 0.0124). Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Cells with DNA fragmentation were present in the ischemic penumbra and injection site after MCAO. (A) Fewer TUNEL-positive cells were detected in rats administered MSC-BDNF than in those administered DMEM (FITC, green, TUNEL-positive; PI, red, nuclear; original magnification ×200) (B) Original magnification ×630. (C) TUNEL-positive cells were significantly reduced in number in animals treated with MSC-BDNF in the ischemic boundary zone compared to animals injected with DMEM (P = 0.013). (D) Fewer positive cells were detected in rats administered MSC-BDNF compared to those that received MSC. Many DsR-positive MSC were detected within <2 mm of the injection site. FITC (green, TUNEL positive), DsR (red, MSC). Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Photomicrographs showing the morphologic features of exogenous MSC and endogenous brain cells in the rat brain. Using double immunofluorescence staining, EGFP cells were localized near the injection site. Laser scanning confocal microscopy showed EGFP cells (green), neuronal nuclear antigen (NeuN; A), and glial fibrillary acidic protein (GFAP; B) in a recipient rat brain. Scale bar, 20 μm. Molecular Therapy 2004 9, 189-197DOI: (10.1016/j.ymthe.2003.10.012) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions