从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞
Summary
The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.
Abstract
成年鼠和人的脊髓神经干/祖在生长因子富集培养基中培养细胞（NSPCs）允许，自我更新的和具有发泡性的神经干细胞的多能增殖。在血清条件下，这些多能NSPCs将分化，产生神经元，星形胶质细胞和少突胶质。所收获的组织被酶促解离以木瓜蛋白酶-EDTA溶液，然后机械地分离，并通过一个不连续密度梯度分离，得到是镀在补充有表皮生长因子（EGF）的Neurobasal培养基，碱性成纤维细胞生长因子的单个细胞悬浮液（bFGF的），和肝素。成年大鼠脊髓NSPCs是生长的贴壁培养的自由浮动的神经球和成人脊髓NSPCs培养。在这些条件下，成人脊髓NSPCs增殖，前体细胞的明确标记，并在通道可连续展开。这些细胞可以bê研究在体外响应于各种刺激，而外部因素可以被用来促进谱系限制检查神经干细胞的分化。多能NSPCs或其后代也可以移植到各种动物模型来评估再生修复。
Introduction
NSPCs致力于能够自我更新和体外易于扩展神经系多能细胞。我们将这些细胞作为神经干/祖细胞的混合群，因为它们显示自我更新的多能干细胞和更限制性祖细胞的性质。 NSPCs被发现在胎儿和成人脑和脊髓1,2-两者。在成人中，NSPCs通常静态和驻留特定龛包括脑室下区衬侧脑室2-4，以及围绕脊髓5,6的中央管室周区域内。
通常情况下，NSPCs如在无血清培养基中自由浮动的神经球或以贴壁单层补充EGF和bFGF，这对于干细胞/祖细胞群选择分裂素中培养。最初由雷诺和Weiss 2开发的神经球测定中，是最常用的培养和扩大的神经干细胞。 NSPCs显示多能的时候都接种于生长因子 – 自由含培养基的血清，分化成神经元，星形胶质细胞和少突胶质。多能，自我更新NSPCs可以分离和培养从成年啮齿动物脊髓当培养组织包括中央管6,7的区域。使用从相对于其他区域产生的细胞在成人脊髓生成NSPCs的潜在的优点是，这些组织特异性细胞最相似的细胞在脊髓被丢失或损坏的以下的损伤或疾病。
以前的工作表明，从成人脊髓衍生神经球可能不会传播长期或传代来产生足够数量的细胞8,9。但是，在培养条件的修改中，我们报道了成人脊髓源性NSPCs膨胀和移植，这表明自我更新和多能NSPCs可以从器官移植供体10的成人脊髓中分离。首先，大多数的白质解剖和这些培养细胞中除去生长因子富含介质粘附基板上选择用于增殖成人脊髓NSPCs。在这个协议中，我们描述了从成年大鼠和人类器官移植的供体，脑室周围组织的解剖，并NSPCs的分离，培养和扩大了脊髓的收获。
Protocol
Representative Results
Discussion
在大鼠脊髓组织护理的解剖应采取在执行椎板切除不要损坏脊髓。这是比较容易分离脑室周围组织时的脊髓节段都完好无损。的组织片段应全部浸入夹层缓冲器和上覆脑膜和白质可以被切掉为的纵向条带microscissors。另外，精细组织钳可以用来剥白质离开。
为隔离程序NSPCs，我们使用了木瓜蛋白酶EDTA解离法结合机械研磨以解离的大鼠和人的脊髓组织。相比于其他蛋白酶，木瓜?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge support from Spinal Cord Injury Ontario, the Ontario-China Research and Innovation Fund, the Toronto General and Western Hospital Foundation, and Physicians’ Services Incorporated Foundation. The authors thank Dr. Tasneem Zahir for expert advice in human spinal cord NSPC culture, and Drs. Cindi Morshead and Iris Kulbatski for their expert advice in rat spinal cord NSPC isolation.
Materials
| 1X PBS | Life Technologies | #10010023 | Dissection buffer |
| 1X HBSS | Life Technologies | #14175095 | Dissection buffer |
| D-glucose | Sigma | # G-6152 | Prepare 30% glucose stock solution for dissection buffer and hormone mix |
| Penicillin-Streptomycin | Life Technologies | #15140-148 | Dissection buffer and culture medium |
| Neurobasal-A | Life Technologies | #10888-022 | Culture medium |
| L-glutamine, 200mM | Life Technologies | #25030-081 | Culture medium |
| B27 | Life Technologies | #12587010 | Culture medium |
| DMEM | Life Technologies | #11885084 | Hormone mix |
| F12 | Life Technologies | #21700-075 | Hormone mix |
| NaHCO3 | Sigma | # S-5761 | Prepare 7.5% NaHCO3 stock solution for hormone mix |
| HEPES | Sigma | #H9136 | Prepare 1M HEPES stock solution for hormone mix |
| Insulin | Sigma | #I-5500 | Hormone mix |
| Apo-transferrin | Sigma | #T-2252 | Hormone mix |
| Putrescine | Sigma | # P7505 | Hormone mix |
| Selenium | Sigma | #S-9133 | Hormone mix |
| Progesterone | Sigma | #P-6149 | Hormone mix |
| EGF, mouse | Sigma | #E4127 | Prepare 100 μg/ml stocks in B27 and aliquot; EFH medium |
| EGF, human recombinant | Sigma | #E9644 | Prepare 100 μg/ml stocks in B27 and aliquot; EFH medium |
| bFGF, human recombinant | Sigma | #F0291 | Prepare 100 μg/ml stocks in B27 and aliquot; EFH medium |
| Heparin, 10000U | Sigma | #H3149 | Prepare 27.3 mg/ml stocks in hormone mix and aliquot; EFH medium |
| Papain dissociation kit | Worthington Biochemicals | #LK003150 | Contains EBSS, papain, DNase, ovomucoid protease inhibitor with BSA |
| Sodium Pentobarbital | Bimeda – MTC Animal Health Inc | DIN 00141704 | |
| Tissue Forceps: Addisons | Fine Science Tools | #11006-12 | Serrated standard tip; micro-tip also available |
| Fine Forceps: Dumont #4 | Fine Science Tools | #11241-30 | |
| Microscissors | Fine Science Tools | #15024-10 | Round-handled Vannas |
| Rongeurs | Bausch & Lomb | N1430 | |
| 10mm Petri dishes | Nunc | 1501 | |
| T25 Culture flasks | Nunc | 156367 | |
| 40 μm nylon cell strainer | VWR | CA21008-949 | |
| 6 well plates | Nunc | CA73520-906 | |
| Matrigel, growth factor reduced (100X) | Corning | 354230 | Thaw according to directions and freeze aliquots; use diluted at a ratio of 40 μl Matrigel: 1 ml SFM |
Referencias
- Gage, F. H. Mammalian neural stem cells. Science. 287, 1433-1438 (2000).
- Reynolds, B. A., Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 255, 1707-1710 (1992).
- Gritti, A., et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci. 16, 1091-1100 (1996).
- Morshead, C. M., et al. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron. 13, 1071-1082 (1994).
- Weiss, S., et al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci. 16, 7599-7609 (1996).
- Martens, D. J., Seaberg, R. M., van der Kooy, D. In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur J Neurosci. 16, 1045-1057 (2002).
- Kulbatski, I., et al. Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization. J Histochem Cytochem. 55, 209-222 (2007).
- Akesson, E., et al. Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres. Physiol Behav. 92, 60-66 (2007).
- Dromard, C., et al. Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro. J Neurosci Res. 86, 1916-1926 (2008).
- Mothe, A. J., Zahir, T., Santaguida, C., Cook, D., Tator, C. H. Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation. PLoS One. 6, e27079 (2011).
- Huettner, J. E., Baughman, R. W. Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci. 6, 3044-3060 (1986).
- Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. J Vis Exp. , (2010).
- Reynolds, B. A., Rietze, R. L. Neural stem cells and neurospheres–re-evaluating the relationship. Nat Methods. 2, 333-336 (2005).
- Singec, I., et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods. 3, 801-806 (2006).
- Louis, S. A., Azari, H., Sharififar, S., Vedam-Mai, V., Reynolds, B. A. Neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells. J Vis Exp. , (2011).
- Louis, S. A., et al. Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay. Stem Cells. 26, 988-996 (2008).
- Conti, L., et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 3, e10 (2005).
- Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex. 16, i112-i120 (2006).
