作者衷心感谢王培acknowledge博士提供的缺氧室仪器，以及Alice Lui女士的技术支持。

所有涉及动物的手续均严格按照NIH"实验动物护理和使用指南"进行，并经香港大学李嘉诚医学院动物实验动物教学与研究委员会批准。获得知情同意后，从健康供体的髂嵴获得人骨髓样品。香港大学机构审查委员会批准议案。 1.大鼠MSC培养物的制备<ol><li> 从股骨收获MSC <ol><li>在使用前，将所有的夹层工具（ 即精细剪切剪刀，钝头切割剪刀和齿形钳子）在180°C下高压灭菌至少2小时。 </li><li>制备包含补充有15％胎牛血清（FBS）和青霉素/链霉素（P / S，1％v / v）的最小必需培养基-α修饰（αMEM）的MSC生长培养基）。 </li><li>用戊巴比妥过量（240mg / kg体重，腹膜内）牺牲年轻的雄性Sprague Dawley大鼠（体重200-250g）。 注意:来自不同大鼠的骨髓样品应分开处理。 </li><li>将牺牲的动物置于仰卧位。用70％乙醇彻底清洁他们的腹部和下肢。 </li><li>使用精细的解剖剪刀和镊子去除内侧大腿上的皮肤和皮下组织。沿周向除去大腿肌肉直到股骨暴露。继续向近端和远端，直到看到膝盖和髋关节。通过髋关节和膝关节脱位股骨，使用钝头切割剪刀。 注意:在这个阶段不要横断股骨以露出骨髓腔。将完整的股骨转移到层流组织培养罩以进一步处理。 </li><li>使用钝头切割剪刀穿过干to端穿过股骨的远端和近端。 </li><li>将70μm的细胞过滤器放在50mL锥形管上。将含有磷酸盐缓冲盐水（PBS，10 mM Na 2 HPO 4 ，pH 7.4）的21 G，10 mL注射器插入裸露的股管，并通过反复冲洗将骨髓内容物冲洗成锥形管。 注意:使用约20 mL PBS冲洗每个股骨。如果被冲洗的内容物的颜色保持血染和混浊，则可以使用较大的体积。 </li><li>通过480×g离心5分钟收集细胞。丢弃上清液。将细胞沉淀重悬于10mL的MSC生长培养基中。将细胞置于10厘米的组织培养皿上。将组织培养皿放入细胞培养箱（37℃，5％CO 2 ）中。将电镀的初始日期记录为第0天。 注意:在涉及离心的所有步骤中，设置最大减速度的制动器。 </li></ol></li><li> 建立和扩建MSC殖民地 注意:本协议r组织培养上的精液塑性粘附作为从骨髓腔内选择MSC的手段11 。允许骨髓含量粘附到组织培养塑料上2天。 <ol><li>在第2天，用10mL PBS冲洗培养板三次以去除非贴壁细胞。冲洗后用10mL的生长培养基替换PBS。用PBS洗涤细胞，每3天补充MSC生长培养基。 注意:MSC殖民地在第6-7天应该可见（ 图2A ）。 </li><li>在第10天通过除去生长培养基并用PBS冲洗细胞，使细胞通过细胞。加入1.5 mL重组酶切细胞解离试剂，37℃孵育5 min。加入3 mL MSC生长培养基中和反应。通过以250×g离心5分钟收集分离的细胞。 </li><li>在PBS中适当稀释后，使用血细胞计数器量化沉淀中的细胞。 </li><li>将种子传代细胞以40,000个细胞/ cm 2的密度转移到MSC生长培养基中的10cm培养板中。 注意:大鼠MSC在传代后2天内应达到80-90％汇合（ 图2B ）。细胞可以如步骤1.2.2所述传代达8代。 MSC可以通过免疫细胞化学和它们的三线分化能力来表征（ 图3 ）。通道3和8之间只有MSC培养物经受随后的缺氧预处理和神经祖细胞富集。更大通道数的MSC采用扁平形态（ 图2C ），不产生足够数量的神经祖细胞。这些文化应该被丢弃。 </li></ol></li></ol> 2.人类BMSC培养物的制备<ol><li>用9 mL MSC生长培养基稀释1 mL人骨髓抽吸液，细胞在10厘米的组织培养皿上。将培养物保持在细胞培养箱（37℃，5％CO 2 ）中。 </li><li> 2天后取出培养基，用10mL PBS轻轻冲洗培养液三次以去除非贴壁细胞。最后冲洗后，取出PBS，并用10 mL的MSC生长培养基更换。在PBS冲洗后每3天培养一次，补充生长培养基。 注意:第6-7天，MSC菌落应该可见。殖民地的数量可能会在受试者之间变化。 </li><li>在第10天通过细胞，如步骤1.2.2所述。在PBS中适当稀释后，使用血细胞计数器量化沉淀中的细胞。将传代细胞以40,000个细胞/ cm 2的密度接种到MSC生长培养基中的10cm培养板上。 注意:人类MSC（ 图2D ）显示与大鼠MSC相似的形态，同样应在传代后2天内达到80-90％汇合。他们应该是chara通过免疫细胞化学和它们的三线分化能力进行调整12 。与大鼠MSC一样，在第3代和第8代之间的人类MSC接受后续的缺氧预处理和神经祖细胞富集。 </li></ol>缺氧预处理<ol><li>拆开环形夹具后，拆除缺氧室部件（ 即底座，盖子，托盘），并用70％乙醇清洁各个部件。将腔室部件置于层流组织培养罩内，在UV光下灭菌15分钟。 </li><li>在缺氧预处理之前，取出培养基，并用10mL PBS冲洗大鼠和人MSC培养物（第1和第2部分）。用10mL补充有25mM HEPES的MSC生长培养基替换PBS。 注意:培养在10厘米盘子上的MSC在进行缺氧预处理前应达到80-90％的汇合度。 </li><li>放cul在缺氧室内的菜肴。重新组装腔室部件并拧紧环夹。将99％N 2 /1％O 2的气体混合物以10升/分钟的流速冲洗到室中5分钟。 </li><li>密封缺氧室的连接端，确保没有气体泄漏。将细胞培养箱（37℃，5％CO 2 ）放置室16小时。 </li><li>完成缺氧预处理后，从室中取出培养物，为后续的神经祖细胞富集培养做准备。 </li></ol>神经祖细胞丰富文化<ol><li>制备由补充有B27（2％v / v），碱性成纤维细胞生长因子（bFGF，20ng / mL），表皮生长因子（EGF，EGF）的Dulbecco修饰的Eagle培养基/ Ham的营养混合物F12（DMEM / F12）组成的神经祖细胞培养基20ng / mL）和P / S（1％v / v）。 </li><li>分离缺氧预处理的大鼠/人类MSC，如步骤1.2.2所述。通过以250×g离心5分钟收集分离的细胞。使用血细胞计数器在PBS中适当稀释后，定量细胞沉淀。 </li><li>将细胞重悬于神经祖细胞培养基中，并以6,000个细胞/ cm 2的密度将其种子置于低附着的6孔板上。将培养物置于细胞培养箱（37℃，5％CO 2 ）中12天。每3天补充75％的神经祖细胞。 注意:在第6-7天应该观察到大小的非粘附细胞簇。在第10-12天，可以观察到直径≥100μm的神经球（ 图4 ）。与在正常氧条件下培养的MSC相比，低氧预处理的MSC应该产生更多的神经球。 </li><li>通过将其吸入10 mL移液管并将其转移到15 mL锥形管中，在第12天收集神经球。离心神经球250 xg 5分钟。 注意:神经球可以特征在第12天用于神经祖细胞标志物，如巢蛋白和GFAP 7 。 </li></ol>通过与DRG神经元的共培养生成命运致命的雪旺氏细胞<ol><li> 制备纯化的大鼠DRG神经元 <ol><li>在使用前，将所有的夹层工具（ 即，夹层剪刀，镊子，两个显微切割钳和显微解剖剪刀）高压灭菌至少2小时。 </li><li>在4℃下，将具有聚-D-赖氨酸（PDL，10μg/ mL PBS）的6孔组织培养板过夜。取出PDL，每孔用1.5 mL PBS冲洗。 </li><li>继续在37℃下用层粘连蛋白（10μg/ mL，PBS）包被2小时。每孔用1.5 mL PBS冲洗平板。 </li><li>制备DRG神经元维持培养基，其中包括补充有B27（2％v / v），L-谷氨酰胺（1％v / v），神经生长因子（NGF，20ng / mL）和P / S（1 ％v / v）。 </li><li>制备DRG神经元纯化培养基，由补充B27（2％v / v），L-谷氨酰胺（1％），NGF（20ng / mL），P / S（1％），氟脱氧尿苷（FDU，10 μg/ mL）和尿苷（10μg/ mL）。 </li><li>通过戊巴比妥过量（240mg / kg体重，腹膜内）在妊娠14-15日怀孕大鼠。 </li><li>将牺牲的动物置于仰卧位。用70％乙醇彻底清洁腹部。 </li><li>使用精细的解剖剪刀和镊子纵向切割动物的下腹壁。使用解剖剪刀识别并清除子宫。切割子宫壁暴露和提取胚胎。将胚胎转移到装有PBS的无菌10cm培养皿中。将培养皿放在冰上。 </li><li>将用于解剖的胚胎转移到装有PBS的无菌10cm培养皿（室温）中并将其置于解剖显微镜下。让胚胎处于俯卧位。 不E:发白的脊髓和附着的DRG可以通过其半透明的皮肤在胚胎的背部看到。 </li><li>沿脊髓的两侧插入微分解钳，并使用钝性剥离开始将脊髓与周围的软组织分开。使用显微解剖镊子沿着颈部开口和尾巴切割没有动物的脊髓。在电线的腹侧进行进一步的钝性剥离，以将其从周围的软组织中释放出来。 </li><li>使用微分解钳去除残留的软组织，在脱离脊髓的背侧。 注意:在这个阶段，只有脊髓，神经根和附着的DRG应该保留。 </li><li>使用显微解剖钳从其连接的神经根分离个体DRG。使用连接到1 mL尖端的移液器笔将DRG转移到含有PBS的1.5 mL无菌离心管中。 注意:对于每个1.5 mL管，最多为可以容纳100个DRG。 </li><li>以250 xg的速度将DRG离心5分钟，并重新悬浮于重组酶切细胞解离试剂（每管200μL）。孵育（37℃，5％CO 2 ）10分钟。以250 xg离心DRG 5分钟，取出上清液，重悬于DRG神经元维持培养基中。通过使用200μL移液管尖端轻轻研磨分离沉淀。在适当稀释后使用血细胞计数器量化沉淀中的细胞。 </li><li>将细胞以5,000个细胞/ cm 2的密度种植在每孔1.5mL的DRG神经元维持培养基中的PDL /层粘连蛋白包被的6孔板中。培养两天后，取出DRG神经元维持培养基，用PBS冲洗，更换为DRG神经元纯化培养基。 注意:对于每个纯化周期，用纯化培养基处理DRG培养物2天，然后在维持培养基中孵育1天。 3-4次纯化循环后，预期所有内源性神经胶质的卵圆形7 。这应该需要大约14天。纯化的培养物对神经元标记TUJ1测试为阳性，并且S100β表达缺失（ 图5 ）。 </li></ol></li><li> 雪旺细胞样细胞的产生 <ol><li>制备补充有β-非调白蛋白（100ng / mL），bFGF（10ng / ml），血小板衍生生长因子（PDGF-AA，5ng / mL），FBS（10％）和P / S（1％v / v）。 </li><li>将第4部分制备的神经球以每孔5-10微米的密度，每孔5-10毫升的神经胶质细胞诱导培养基在PDL /层粘连蛋白包被的6孔板中制备。用PBS清洗细胞后，每2天更换一次胶质细胞诱导培养基。 注意:接种的神经球的细胞被认为在第2天向外迁移。到第7天，迁移细胞具有锥形外观，并应证明施万细胞的免疫阳性标志物p75神经营养因子受体（p75）和S100β7。这些细胞被称为SCLC。 </li></ol></li><li> SCLCs与DRG神经元的共培养 <ol><li>以1:1体积与体积比制备由DRG神经元维持培养基（步骤5.1.4）和胶质诱导培养基（步骤5.2.1）组成的共培养培养基。 </li><li>准备由补充有FBS（5％），β-非调白蛋白（10ng / mL）和P / S（1％v / v）的DMEM / F12组成的施万细胞维持培养基。 </li><li>从第7天SCLC中取出培养基，用PBS冲洗，并将其与0.5mL /孔的重组酶切细胞解离试剂在37℃孵育5分钟。将SCLC重悬于共培养培养基中。 </li><li>在适当稀释后用血细胞计数器量化细胞。 </li><li>将SCLC以1,000个细胞/ cm 2的密度接种到纯化的DRG神经元培养物上。保持共培养14天，每2天更换一次。 注意:在共培养期间，SCLC获得了成熟的施万细胞典型的纺锤状形态（ 图6 ）。这些细胞在生长因子撤出后仍然保持其表型，并且能够在体外和体内髓鞘髓鞘7,10。施万细胞标志物（ 即 p75和S100β）的阳性应通过免疫荧光进行监测。 </li><li>共同培养完成后，通过命运提出的施万细胞，如步骤1.2.2所述。每孔使用0.5 mL离解试剂。在适当稀释后用血细胞计数器量化细胞。 </li><li>以10,000个细胞/ cm 2的密度重悬在施万细胞维持培养基中的命运承诺的雪旺氏细胞。将种子细胞分成PDL /层粘连蛋白包被的6孔板进行免疫荧光。 注意:共培养不可避免地含有已经采用成纤维细胞的MSC细胞命运7 。在共同培养完成后，这些细胞与命运致命的施万细胞一起传播。位于该成纤维细胞基质顶部的雪旺氏细胞可以容易地分离，并且在PBS（4℃）的"冷喷"中冲洗后进一步扩大，如其他地方所述。命运承诺的施万细胞可以在维持培养基中扩增1个月。 </li></ol></li></ol>

<ol>
	<li>Wiliams, R. R., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation:+a+repair+strategy+for+spinal+cord+injury?.">Schwann cell transplantation: a repair strategy for spinal cord injury?.</a> Prog Brain Res. 201, 295-312 (2012).</li><li>Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation+for+spinal+cord+injury+repair:+its+significant+therapeutic+potential+and+prospectus.">Schwann cell transplantation for spinal cord injury repair: its significant therapeutic potential and prospectus.</a> Rev Neurosci. 26 (2), 121-128 (2015).</li><li>Lindvall, O., Kokaia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+human+neurodegenerative+disorders--time+for+clinical+translation?.">Stem cells in human neurodegenerative disorders--time for clinical translation?.</a> J Clin Invest. 120 (1), 29-40 (2010).</li><li>Terzic, 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=Directed+Differentiation+of+Oligodendrocyte+Progenitor+Cells+From+Mouse+Induced+Pluripotent+Stem+Cells.">Directed Differentiation of Oligodendrocyte Progenitor Cells From Mouse Induced Pluripotent Stem Cells.</a> Cell Transplant. 25 (2), 411-424 (2016).</li><li>Takashima, Y., 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=Neuroepithelial+cells+supply+an+initial+transient+wave+of+MSC+differentiation.">Neuroepithelial cells supply an initial transient wave of MSC differentiation.</a> Cell. 129 (7), 1377-1388 (2007).</li><li>Felling, R. J., 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=Neural+stem/progenitor+cells+participate+in+the+regenerative+response+to+perinatal+hypoxia/ischemia.">Neural stem/progenitor cells participate in the regenerative response to perinatal hypoxia/ischemia.</a> J Neurosci. 26 (16), 4359-4369 (2006).</li><li>Shea, G. K., Tsui, A. Y., Chan, Y. S., Shum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+Schwann+cells+achieve+fate+commitment--a+prerequisite+for+remyelination+therapy.">Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.</a> Exp Neurol. 224 (2), 448-458 (2010).</li><li>Jessen, K. R., Mirsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+development+of+glial+cells+in+peripheral+nerves.">The origin and development of glial cells in peripheral nerves.</a> Nat Rev Neurosci. 6 (9), 671-682 (2005).</li><li>Mung, K. 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=Rapid+and+efficient+generation+of+neural+progenitors+from+adult+bone+marrow+stromal+cells+by+hypoxic+preconditioning.">Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.</a> Stem Cell Res Ther. 7 (1), 146 (2016).</li><li>Ao, Q., 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=The+regeneration+of+transected+sciatic+nerves+of+adult+rats+using+chitosan+nerve+conduits+seeded+with+bone+marrow+stromal+cell-derived+Schwann+cells.">The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells.</a> Biomaterials. 32 (3), 787-796 (2011).</li><li>Tondreau, T., 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=Isolation+of+BM+mesenchymal+stem+cells+by+plastic+adhesion+or+negative+selection:+phenotype,+proliferation+kinetics+and+differentiation+potential.">Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.</a> Cytotherapy. 6 (4), 372-379 (2004).</li><li>Baksh, D., Song, L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells:+characterization,+differentiation,+and+application+in+cell+and+gene+therapy.">Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.</a> J Cell Mol Med. 8 (3), 301-316 (2004).</li><li>Jirsova, K., Sodaar, P., Mandys, V., Bar, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+jet:+a+method+to+obtain+pure+Schwann+cell+cultures+without+the+need+for+cytotoxic,+apoptosis-inducing+drug+treatment.">Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.</a> J. Neurosci. Methods. 78 (1-2), 133-137 (1997).</li></ol>

本手稿的所有作者均无披露声明。

在通过缺氧预处理和神经球培养富集神经祖细胞之前，必须保持MSC的"干性"。根据我们的经验，可以通过其细长的成纤维细胞样形态可靠地鉴定多能MSC。相比之下，采用更扁平，四边形形态，具有突出的细胞骨架应力纤维的MSC不容易采用神经细胞命运，应该被丢弃。一般来说，我们不使用通道数大于8的MSC。为了保持它们的干性，在达到100％汇合之前及时通过MSC是至关重要的。相反，保持MSC在一个?...

神经祖细胞及其衍生物的移植显示出创伤神经损伤1,2和神经变性3,4之后的治疗策略的希望。在临床应用之前，必须确保:i）获取和扩大自体来源的干/祖细胞的方法，以及ii）将其引导到相关的成熟细胞类型的手段3 。我们对细胞治疗脊髓损伤的兴趣导致我们从成人组织中寻找一个健壮的自体细胞来源的神经祖细胞。 MSCs的亚群源自神经嵴，并且容易从骨髓腔进入。这些细胞是可以产生神经元和神经胶质的神经祖细胞5 。脑缺血的动物模型表明，缺氧促进了唾液<html>脑内神经祖细胞的多发性和多能性6 。这是利用缺氧预处理作为扩展骨髓来源的神经祖细胞的手段的基础。 将施万细胞移植到损伤的脊髓中促进再生2 。 SCLC可以通过补充Gliogenic因子（ 即， β-Heregulin，bFGF和PDGF-AA）从MSC产生，但表现出表型不稳定性。退出生长因子后，它们恢复成为成纤维细胞样表型7 。由于异常分化和致癌的风险，细胞移植中的表型不稳定性是不合需要的。由于施旺细胞前体与胚胎周围神经8内的轴突束相关，我们被引导与纯化的胚胎DRG神经元共培养SCLC 7 ，ass ="xref"&gt; 9。结果成熟的雪旺氏细胞是命运决定的，并在体外证明功能7,9和体内 10 。 我们用于富集MSC的神经祖细胞的方案是简单和有效的，导致后续测定的细胞数量增加。通过共培养平台推导命运致命的雪旺氏细胞允许研究胶质细胞分化和产生用于潜在临床应用的稳定和功能的施万细胞。

<table><tbody><tr><td>&amp;alpha;MEM</td><td>Sigmaaldrich</td><td>M4526</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermofisher scientific</td><td>12400-024</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher scientific</td><td>21103-049</td><td></td></tr><tr><td>FBS</td><td>Biosera</td><td>FB-1280/500</td><td></td></tr><tr><td>B27</td><td>Thermofisher scientific</td><td>17504-001</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>Thermofisher scientific</td><td>PHG0313</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)&amp;nbsp;</td><td>Peprotech</td><td>100-18B/100UG</td><td></td></tr><tr><td>Nerve growth factor (NGF)&amp;nbsp;</td><td>Millipore</td><td>NC011</td><td></td></tr><tr><td>Platelet-derived growth factor-AA (PDGF-AA)</td><td>Peprotech</td><td>100-13A</td><td></td></tr><tr><td>Heregulin beta-3, EGF domain (&amp;beta;-Her)</td><td>Millipore</td><td>01-201</td><td></td></tr><tr><td>Uridine</td><td>Sigmaaldrich</td><td>U3003</td><td></td></tr><tr><td>5-Fluro-2&#039; - deoxyuridine (FDU)</td><td>Sigmaaldrich</td><td>F0503</td><td></td></tr><tr><td>Poly-D-lysine (PDL)</td><td>Sigmaaldrich</td><td>P7886-1G</td><td></td></tr><tr><td>Laminin</td><td>Thermofisher scientific</td><td>23017015</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermofisher scientific</td><td>35050061</td><td></td></tr><tr><td>Penicillin / streptomycin (P/S)</td><td>Thermofisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>TrypLE Express</td><td>Thermofisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>10 cm plate for adherent culture</td><td>TPP</td><td>93100</td><td>Used for selection of MSCs by tissue culture adherence</td></tr><tr><td>6-well plate for adherent culture</td><td>TPP</td><td>92006</td><td>Used for expansion of MSCs following passaging</td></tr><tr><td>UltraLow 6-well plate for non-adherent culture</td><td>Corning</td><td>3471</td><td>Used for neural progenitor enrichment</td></tr><tr><td>anti-human CD90(Thy-1)</td><td>BD Biosciences</td><td>555593</td><td></td></tr><tr><td>anti-human CD73</td><td>BD Biosciences</td><td>550256</td><td></td></tr><tr><td>anti-human/rat STRO-1</td><td>R&amp;amp;D Systems</td><td>MAB1038</td><td></td></tr><tr><td>anti-human nestin</td><td>R&amp;amp;D Systems</td><td>MAB1259</td><td></td></tr><tr><td>anti-human CD45</td><td>BD Biosciences</td><td>555480</td><td></td></tr><tr><td>anti-rat CD90(Thy-1)</td><td>BD Biosciences</td><td>554895</td><td></td></tr><tr><td>anti-rat CD73</td><td>BD Biosciences</td><td>551123</td><td></td></tr><tr><td>anti-rat nestin</td><td>BD Biosciences</td><td>MAB1259</td><td></td></tr><tr><td>anti-rat CD45</td><td>BD Biosciences</td><td>554875</td><td></td></tr><tr><td>Anti-S100&amp;beta;</td><td>Dako</td><td>Z031101</td><td></td></tr><tr><td>Anti-p75</td><td>Millipore</td><td>MAB5386</td><td></td></tr><tr><td>Anti-GFAP</td><td>Sigmaaldrich</td><td>G3893</td><td></td></tr><tr><td>Anti-Class III-beta tubulin (Tuj-1)</td><td>Covance</td><td>MMS-435P</td><td></td></tr><tr><td>Anti-Human nuclei</td><td>Millipore</td><td>MAB1281</td><td></td></tr><tr><td>Hypoxia chamber</td><td>Billups-Rothenberg</td><td>MIC-101</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigmaaldrich</td><td>H4034-100G</td><td></td></tr></tbody></table>

hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

该手稿描述了一种从骨髓基质细胞（MSC）群体中丰富神经祖细胞并随后将其引导至成熟的施万细胞命运的手段。我们将大鼠和人类MSC接受短暂的缺氧条件（1％氧气16小时），随后在含有表皮生长因子（EGF）/碱性成纤维细胞生长因子（bFGF）补充的低附着底物上扩增为神经球。将神经球接种到聚-D-赖氨酸/层粘连蛋白包被的组织培养塑料上，并在含有β-Heregulin，bFGF和血小板衍生生长因子（PDGF）的胶质细胞混合物中培养以产生施旺细胞样细胞（SCLC）。 SCLC通过共同培养2周，以E14-15怀孕的Sprague Dawley大鼠获得的纯化的背根神经节（DRG）神经元为指导。成熟的雪旺氏细胞表现出S100β/ p75表达的持续性，并可形成髓鞘细胞。以这种方式生成的细胞具有潜力脊髓损伤后自体细胞移植中的应用，以及疾病建模。

我们的协议关键阶段的概述如图1所示 。总之，通过依靠组织培养塑料选择大鼠和人类MSC。扩张的MSC被缺氧预处理，然后经历神经球形成条件。电镀神经球，使其分化成SCLC。 SCLC与纯化的DRG神经元共培养以产生命运赋予的施旺细胞。 培养的大鼠和人类MSC的形态如图2所示 。显示出与?...

Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

具有神经电位的骨髓基质细胞（MSC）存在于骨髓内。我们的方案通过缺氧预处理丰富了这种细胞群，之后引导他们成为成熟的雪旺氏细胞。

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

hypoxic-preconditioning-marrow-derived-progenitor-cells-as-source-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  The University of Hong Kong. 具有神经电位的骨髓基质细胞（MSC）存在于骨髓内。我们的方案通过缺氧预处理丰富了这种细胞群，之后引导他们成为成熟的雪旺氏细胞。 

视频: Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

从E13小鼠胶质限制前体的推导

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

科研

神经科学

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

免疫与感染

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

生物学

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

生物工程

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

摘要

探索更多视频

此视频中的章节

从E13小鼠胶质限制前体的推导

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

表型分析和分离小鼠造血干细胞和传承的承诺祖

准备可调细胞外基质微环境，以评估施万细胞表型规范

小鼠骨髓移植程序研究克隆造血症

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用