This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

按照新南威尔士，澳大利亚的法律， 验尸脑组织从对安乐死无关的研究的理由成年犬收购。 1.培养基的制备<ol><li>通过加入0.1克明胶粉末到100毫升蒸馏水中并在37℃下搅拌直至溶解制备0.1％的明胶溶液。消毒通过UV照射该溶液15分钟。然后，该媒体可以被存储在4℃下长达1个月。 </li><li>通过加入167毫升F-12的至500ml的DMEM（4.5 g / L的D-葡萄糖）的Dulbecco改良的Eagle培养基（DMEM）和营养混合物F12的比例为1:制备3。加6.7毫升的青霉素/链霉素溶液（为1％的最终浓度）。此媒体可存储在黑暗中在4℃下长达1个月。 </li><li>通过组合440毫升的DMEM（4.5 g / L的D-葡萄糖）中，制成500毫升之血清培养基5毫升的L-丙氨酰-L-谷氨酰胺二肽（200毫米），将5ml青霉素/链霉素和50毫升胎儿BOVIN的Ë血清（FBS）。此媒体可存储在黑暗中在4℃下长达1个月。 </li><li>完全生长培养基由神经干细胞（NSC）基础培养基和增殖神经补充-A（NS-A），2％FBS，2微克/毫升肝素，20毫微克/毫升表皮生长因子（EGF）和10毫微克/毫升碱性成纤维细胞生长因子（bFGF）。使此媒体鲜，紧接大脑解剖，性温至37℃的水浴中。 </li></ol> 2.脑提取<ol><li>用碘伏消毒狗的头部背侧面开始在6个小时的验尸提取。 </li><li>使用手术刀，使一个纵向切口沿矢状中线，穿过头盖骨的整个背侧表面，以暴露底层咬肌肌肉。该切口的长度将依赖于狗的种类和年龄。 </li><li>在头盖骨的喙和尾部的边界，使额外的5 - 双方10厘米垂直切割，通进入眼睛和耳朵后面的分别，让皮肤得到充分的体现。 </li><li>使用手术刀，从他们的附件中的咬肌分开，颅骨矢状嵴和刮去头盖骨任何剩余的结缔组织。 </li><li>使用一个振荡骨锯切割颅骨在周线的帽。用探针或手术刀断绝与硬脑膜任何剩余的附件，然后小心地取出颅盖，露出大脑的背水面。 注意:骨的厚度在颅骨不同地区的不同而不同，所以必须谨慎，以不渗透太深，损害底层的大脑。 </li><li>通过将上部颈椎之间手术刀切断脊髓。 </li><li>用一只手轻轻抬起脑，用解剖刀小心释放从颅窝脑和断绝位于其腹侧连接神经和血管。轻轻抬起的B雨出颅骨并进入DPBS的容器。 注意:犬脑的大嗅球可以深深延伸到前颅窝。必须小心除去脑时，应考虑以保留这些结构。 </li></ol> 3.海马解剖<ol><li>冲洗在Dulbecco氏磷酸盐缓冲盐水（DPBS）大脑以去除任何血液，并将其放置背侧向下到150毫米的培养皿。 </li><li>慢慢通过中间矢状平面利用湿式脑刀和利用所述刀片的整个长度的单个切片纵向平分大脑。不要施加额外的下行压力，或使用锯切运动，因为这样可能会损坏组织。 </li><li>放置每个半球内侧表面上，解剖出使用解剖刀和细镊子海马，并将其转移到35毫米组织培养皿中。 </li></ol> 4.隔离神经前体细胞<ol><li>切碎的T使用手术刀刀片成约1mm 3个问题样本。 </li><li>转移组织入15ml管中，加2ml的0.1％胰蛋白酶的EDTA，和在37℃的水浴中孵育7分钟。 </li><li>停止加入4毫升血清培养基的管中的酶反应。沉淀通过离心悬浮液在100×g离心7分钟，除去上清液。 </li><li>重悬浮沉淀在300微升DPBS，并轻轻吹打上下，用1000微升枪头，直到光滑匀浆形式机械分离。 </li><li> 14毫升血清培养基添加到管，并通过一个40微米的细胞滤网通过细胞悬浮液。沉淀通过离心悬浮液在100×g离心7分钟，除去上清液，并在完全生长培养基中重悬。 </li></ol> 5.神经球文化<ol><li>从在分离获得细胞悬浮液，用台盼蓝染料排除和HEMO计数存活细胞的数目流式细胞仪。 </li><li>稀释在完全生长培养基和种子中的细胞悬浮液以1×10 5个细胞/ cm 2到6孔板的未涂覆的孔中。 </li><li>孵育在37℃，5％CO 2和&gt; 95％的湿度为14 - 28天更换50％的生长培养基的每7天。定期测量神经球的直径。如果允许超越100微米增长直径，细胞死亡和自发分化可在神经球内发生。 </li></ol> 6.神经球通道<ol><li>到通道作为浮动培养，当神经球达到直径约100微米，结合所有的含神经球媒体到单个管中。离心在100×g离心7分钟沉淀并去除上清。 </li><li>重悬沉淀在1毫升0.1％胰蛋白酶的EDTA中，并在37℃的水浴中孵育7分钟。轻轻吸取上下100次，用200μl移液管尖，以确保dissociati在神经球。 </li><li>停止在100 xg离心加入5毫升血清培养基的管中，离心7分钟的酶反应。 </li><li>除去上清液，重悬在1ml的细胞沉淀完全生长培养基中并计数使用台盼蓝染料排除和血球存活细胞的数量。 </li><li>稀释在完全生长培养基和种子中的细胞悬浮液以1×10 5个细胞/ cm 2到6孔板的未涂覆的孔中。孵育在37℃，5％CO 2和&gt; 95％的湿度为14 - 28天更换50％的生长培养基的每7天。 </li></ol>神经前体细胞7.贴壁培养<ol><li>添加足够量的0.1％明胶溶液覆盖在培养箱组织培养皿和固化的表面，在37℃进行1小时处理神经球为贴壁培养之前。 </li><li>从浮神经球文化，将所有媒体到一个单管。沉淀neurospheres离心100×g离心7分钟，除去上清液。 </li><li>重悬沉淀在1毫升0.1％胰蛋白酶的EDTA中，并在37℃的水浴中孵育7分钟。轻轻移液器上下100次，用200μl的枪头。 </li><li>加入5毫升血清培养基以制止在100×g离心7分钟的酶反应和离心机。弃去上清，重悬在1ml完全生长培养基将细胞沉淀，并计算使用台盼蓝染料排除和血球存活细胞的数量。 </li><li>稀释在完全生长培养基将细胞悬液，除去从组织培养瓶中的明胶和以1×10 4个细胞/ cm 2接种细胞。 </li><li>孵育细胞在37℃，5％的CO 2和&gt; 95％的湿度。直到培养达到约80％汇合（后约7天）用新鲜，温暖，完整的生长介质更换介质每三天。 </li></ol> 8. Neur人集落形成实验<ol><li>结合神经细胞集落形成细胞（NCFC）测定媒体组件:NCFC无血清培养基，细胞增殖的NS-A，和胶原溶液（3毫克/毫升）。补充本媒体用2微克/ ml肝素，20纳克/毫升的EGF和10ng / ml的bFGF。 </li><li>以下海马组织离解重悬在神经集落形成测定培养基中的细胞，预先加热到37℃的水浴中。种子的细胞以1.1×10 5个细胞/ ml的克隆密度为35毫米的培养皿中。 </li><li>孵育在此半固体胶原基质的细胞在37℃，5％的CO 2和&gt; 95％的湿度。培养28天，取代50％的生长培养基的每7天。 </li></ol> 9.分化神经前体细胞<ol><li>在37℃下处理细胞不同点之前添加5 微克 /平方厘米的层粘连蛋白溶液的足够体积，以覆盖在培养箱组织培养皿和固化的表面24小时化。 注意:删除层粘连蛋白溶液和冲洗两次涂覆表面在DPBS之前立即播种。 </li><li>传代时，种子在DMEM-F12以1×10 4个细胞/ cm 2的细胞悬浮液（3:1）补充有20毫微克/毫升EGF和40毫微克/ ml的bFGF到介质（预热至37℃）层粘连蛋白涂层的培养皿。 </li><li>孵育细胞在37℃，5％的CO 2和&gt; 95％的湿度，并允许细胞增殖3天。 </li><li>区分粘附神经前体细胞，3天后更换分化培养基含有DMEM-F12介质（3:1）介质预加热到37℃，在补充有10纳克源性神经营养因子（BDNF）/ ml的脑。 </li><li>在分化更换50％的培养基每3天。 28天 - 分化在大约21逐渐发生的。 注意:一旦细胞已开始分化，仅在一个钛更换介质的50％我如暴露于空气中可对细胞的健康产生不利影响。 </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

这里描述的协议优化，保持最大限度地提高细胞活力良好的培养条件。期间提取，分离和扩增所采取的速度和护理是至关重要的。建立单层贴壁扩张的一个关键步骤是初级神经球的有效分离。下面这段话，充分游离神经球可能产生二次浮动的神经球。期间媒体的变化，这些神经球可以被移除，解离和重新接种于贴壁单层通道。相反，如果解离后的细胞存活率为80％以下，这可能离解过程中是指示?...

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

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			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
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		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
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			<td height="21" style="height:21px;">24 well culture plate</td>
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			<td style="width:252px;">662160</td>
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			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
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			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
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			<td style="width:252px;">351146</td>
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			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
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			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
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			<td height="21" style="height:21px;">Collagen solution</td>
			<td>Stem Cell Technologies</td>
			<td>04902</td>
			<td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&#160;</td>
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			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
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			<td style="width:252px;">14190250</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
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			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
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			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
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			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
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			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
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			<td style="width:252px;">35050061</td>
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			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
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			<td height="21" style="height:21px;">Laminin (mouse)</td>
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			<td style="width:252px;">23017015</td>
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			<td height="21" style="height:21px;">NCFC serum free medium (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td>5720</td>
			<td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&#160;</td>
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			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
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			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
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			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
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isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

通过使用体外神经前体测定法，神经发生和神经前体细胞群进行了表征，并在整个成年犬海马背腹轴比较。从孤立的海马组织的神经前体细胞形成的内隔离14天浮动的神经球，达到了直径为100微米由28天文化。从背部和腹部分离衍生神经球表现出平均尺寸没有差别了，下面酶解成单个细胞，可以传代浮动的神经球文化。从两个海马区次要浮动神经球能够形成内通道5?...

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

犬大脑是在研究成年神经宝贵的典范。这里介绍的是隔离，并从原发性脑组织扩大成年犬海马神经前体细胞协议。

分离和成人犬海马神经前体扩张

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Research

JoVE Journal

Developmental Biology

7.0K 次观看.  University of Sydney. 犬大脑是在研究成年神经宝贵的典范。这里介绍的是隔离，并从原发性脑组织扩大成年犬海马神经前体细胞协议。 

视频: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

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.

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

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...