Abstract
Aim:
The goal of this project was to develop a rat model for neural stem cell (NSC) transplantation studies in which NSCs were modified with brain-derived neurotrophic factor (BDNF) genes that may permit extensive and reliable analysis of the transplants.
Methods:
NSCs were cultured and purified by limiting dilution assay in vitro and infected with recombinant retrovirus pLXSN-BDNF (BDNF-NSCs) and retrovirus pLXSN (p-NSCs). The expression of BDNF genes in transgenic and control NSC groups was measured by FQ-PCR and ELISA assays. NSCs were then transplanted into the subretinal space of normal rat retinas in four groups, which included NSCs alone, BDNF-NSCs, phosphate buffered saline (PBS) control, and normal control. Survival, migration, and differentiation of donor cells in host retinas were observed with optical coherence tomography (OCT), Heidelberg retina angiograph (HRA), and immunohistochemistry, respectively.
Results:
The results obtained by FQ-PCR demonstrated that the copy numbers of BDNF gene templates from BDNF-NSCs were the highest among the four groups (P<0.05). Consistent with the results of FQ-PCR, BDNF protein level from the supernatant of the BDNF-NSCs group was much higher than that of the other two groups (P<0.05) as suggested by the ELISA assays. HRA and OCT showed that graft cells could successfully survive. Immunohistochemical analysis revealed that transplanted BDNF-NSCs could migrate in the host retinas and differentiate into glial cells and neurons three months after transplantation.
Conclusion:
BDNF promotes NSCs to migrate and differentiate into neural cells in the normal host retinas.
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References
Temple S . Division and differentiation of isolated CNS blast cells in microculture. Nature 1989; 340: 471–3.
Cattaneo E, McKay R . Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature 1990; 347: 762–5.
Gouras P, Du J, Kjeldbye H, Yamamoto S, Zack DJ . Long-term photoreceptor transplants in dystrophic and normal mouse retina. Invest Ophthalmol Vis Sci 1994; 35: 3145–53.
Algvere PV, Berglin L, Gouras P, Sheng Y, Kopp ED . Transplantation of RPE in age-related macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch Clin Exp Ophthalmol 1997; 235: 149–58.
Wan J, Zheng H, Chen ZL, Xiao HL, Shen ZJ, Zhou GM . Preferential regeneration of photoreceptor from Müller glia after retinal degeneration in adult rat. Vision Res 2008; 48: 223–34.
Ourednik V, Ourednik J, Park KI, Teng YD, Aboody KA, Auguste KI, et al. Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS. Novartis Found Symp 2000; 231: 242–62; discussion 262–9, 302–6.
Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol 1998; 16: 1033–9.
Chacko DM, Rogers JA, Turner JE, Ahmad I . Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat. Biochem Biophys Res Commun 2000; 268: 842–6.
Ahmed S, Reynolds BA, Weiss S . BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 1995; 15: 5765–78.
Kamei N, Tanaka N, Oishi Y, Hamasaki T, Nakanishi K, Sakai N, et al. BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures. Spine 2007; 32: 1272–8.
Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003; 422: 688–94.
Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D . Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 1999; 208: 166–88.
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003; 63: 5821–8.
Li HY, Yuan HP, Zhou XR, Shao ZB, Zhou XM . The expression of recombinant retrovirus pLXSN-brain-derived neurotrophic factor in mouse embryonic cell. Chin J Ocular Fundus Dis 2007; 23: 207–8. Chinese.
Zhou XM, Yuan HP, Zhou XR, Qu W, Li HY, Shao ZB . Comparison of efficacy of two purified methods for neural stem cells from the subependymal zone of embryonic rat brain. Rec Adv Ophthalmol 2009; 29: 90–3. In Chinese.
Kodama Y, Hida H, Jung CG, Baba H, Isono M, Kobayashi H, et al. High titer retroviral gene transduction to neural progenitor cells for establishment of donor cells for neural transplantation to parkinsonian model rats. Neurol Med Chir (Tokyo) 2004; 44: 344–51; discussion 352.
Lu P, Jones LL, Snyder EY, Tuszynski MH . Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003; 181: 115–29.
Brüstle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKay RD . In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci USA 1997; 94; 14809–14.
Lundberg C, Field PM, Ajayi YO, Raisman G, Björklund A . Conditionally immortalized neural progenitor cell lines integrate and differentiate after grafting to the adult rat striatum. A combined autoradiographic and electron microscopic study. Brain Res 1996; 737; 295–300.
Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J . Multipotent progenitor cells in the adult dentate gyrus. J Neurobiol 1998; 36: 249–66.
Suhonen JO, Peterson DA, Ray J, Gage FH . Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature 1996; 383: 624–7.
Pease ME, McKinnon SJ, Quigley HA, Kerrigan-Baumrind LA, Zack DJ . Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma. Invest Ophthalmol Vis Sci 2000; 41: 764–74.
Perez MT, Caminos E . Expression of brain-derived neurotrophic factor and of its functional receptor in neonatal and adult rat retina. Neurosci Lett 1995; 183: 6–9.
Ko ML, Hu DN, Ritch R, Sharma SC, Chen CF . Patterns of retinal ganglion cell survival after brain-derived neurotrophtc factor administration in hypertensive eyes of rats. Neurosci Lett 2001; 305; 139–42.
Suzuki T, Ooto S, Akagi T, Amemiya K, Igarashi R, Mizushima Y, et al. Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina. Biochem Biophys Res Commun 2003; 309: 843–7.
Bushman FD . Retroviral integration and human gene therapy. J Clin Invest 2007; 117: 2083–6.
Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986; 6: 2895–902.
Qiu G, Seiler MJ, Mui C, Arai S, Aramant RB, de Juan E Jr, et al. Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats. Exp Eye Res 2005; 80: 515–25.
Cong L, Sun D, Zhang Z, Jiao W, Rizzolo LJ, Peng S . A novel rabbit model for studying RPE transplantation. Invest Ophthalmol Vis Sci 2008; 49: 4115–25.
Choi KC, Yoo DS, Cho KS, Huh PW, Kim DS, Park CK . Effect of single growth factor and growth factor combinations on differentiation of neural stem cells. J Korean Neurosurg Soc 2008; 44: 375–81.
Acknowledgements
This study was supported by a grant from the National Natural Science Foundation of China (No 30471844).
The authors thank Harbin Veterinary Research Institute (HVRI, China Agricultural Academy of Science, CAAS) for providing the experimental location and technical assistance for FQ-PCR.
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Zhou, Xm., Sun, Jb., Yuan, Hp. et al. A rat model for studying neural stem cell transplantation. Acta Pharmacol Sin 30, 1496–1504 (2009). https://doi.org/10.1038/aps.2009.151
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DOI: https://doi.org/10.1038/aps.2009.151
