Name: high throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies
Uploaded: 2015-01-04T14:00:00.000+00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies at JoVE.com

Funding for this research was provided by the US Army Medical Research and Materiel Command (Grant account no. W81XWH-11-1-0700) and the Stem Cell Research Fund. PAB and EMP were recipients of Ames Laboratory Summer Undergraduate Laboratory Internships (SULI).

1.基板制备的96孔板<ol><li>创建的96孔板，概述了不同底物和细胞类型的地图要被检查（ 图1）。 </li><li>得到储备溶液不同的基板〔聚-L-赖氨酸，纤连蛋白，I型胶原，层粘连蛋白，巢蛋白和胶原IV-层粘连蛋白（ECL）]，在96孔多孔板，并准备一个工作台在无菌细胞培养物的引擎盖。 </li><li>通过在无菌磷酸盐缓冲盐水（PBS）稀释的库存为5微克/毫升的最终浓度（基于用于细胞的生长和增殖的基质浓度依赖性测定预先决定该浓度）制备个别基板。混合使用涡流倒入无菌贮存器之前。 </li><li>加入100微升底物溶液导入各孔中，根据所述96孔图谱（ 图1）（12或8通道微量便于micropipetting到96孔板）。密封盖子的96孔板用封口膜条和隔夜存放于4℃。 </li></ol> 2.细胞电镀和时间推移成像注:鼠标的MSC从成年C57BL / 6小鼠的骨髓中分离，并保持作为贴壁细胞系。利用慢病毒载体的编码BDNF（LV-BDNF;和神经胶质细胞源性神经营养因子（人cDNA GDNF）;干细胞用慢病毒载体改造它们分泌脑源性神经营养因子（人cDNA BDNF）感染CMV-BDNF-IRES -GFP），GDNF（LV-GDNF; CMV-GDNF-IRES-GFP）和绿色荧光蛋白（GFP，LV-GFP; CMV-GFP）。 注:培养基为小鼠间充质干细胞（MSCs）是Iscove氏改良的Dulbecco氏培养基含有10％杂交瘤合格的胎牛血清，10％马血清，2mM的L-谷氨酰胺，和10000 U / ml的青霉素，10mg / ml的链霉素。五种不同类型的小鼠的MSCs（MSCS，GFP-的MSC，BDNF-GFP-的MSC，GDNF-GFP-M的种姓和BDNF / GDNF-GFP-的MSCs）接种在T75细胞培养瓶约30％汇合。 <ol><li> 细胞电镀 <ol><li>第二天，除去通过抽吸基片的解决方案，并与大约200微升无菌PBS冲洗井每两次。最终的PBS漂洗后添​​加细胞培养基（200μl/孔）到每个孔中。将96孔板入细胞培养孵化器设置在37℃和5％CO 2的平衡。 </li><li>而在96孔板被平衡在孵化器，收获细胞（在T75烧瓶的MSC应该大约70％汇合时收获/电镀时间）通过收集生长培养基从T75烧瓶（被称为条件培养基），并存储在无菌条件下的15毫升锥形管中（此条件培养基将在下面步骤2.1.4使用）。 </li><li>加入8毫升无菌PBS中至烧瓶中并轻轻摇动，然后移液器关闭PBS中，并加入1 ml 0.05％胰蛋白酶和0.01％EDTA溶液从T75烧瓶的培养表面分离细胞。通过使用倒置显微镜配备相衬光学观看烧瓶监测细胞脱离。 </li><li>当细胞已经分离时，立即加入8毫升条件培养基（收集步骤2.1.2）的烧瓶中。收集的细胞悬浮液，并在450×g离心转移到15毫升锥形离心管中，离心4分钟以沉淀细胞。 </li><li>除去上清液和重悬细胞沉淀在200微升新鲜和温热（37℃）的细胞培养介质。 </li><li>通过使用血细胞计数器进行锥虫蓝活细胞计数测定细胞中的细胞悬浮液的数量。板中的细胞以大约300个细胞/孔的密度向96孔板的适当孔中。 </li><li>重复这些步骤细胞的每个群体。 </li></ol></li><li> 时间推移成像 <ol><li>一旦所有的干细胞都被镀，地点的96孔板进培养箱2小时，以使干细胞附着于基底上。 </li><li>启动HCS系统和等待2小时，以便系统平衡。设置环境控制器至37℃，并连接含有5％的CO 2在空气中的HCS系统环境室供给一个恒定的空气源的混合气体钢瓶。 </li><li>从培养箱以下的2小时的平衡期取出96孔板，并直接放置到HCS系统的细胞生长室。允许30分钟以达到平衡，以占该板的任何热相关的膨胀，然后启动图像采集和分析软件来配置该板设置。 </li><li>选择20X客观的成像。选择[ 即，GFP-的MSC上纤连蛋白等。（6基板×5的MSC亚型为总共30条件式两次= 60井总数）]用于设置延时成像每种条件两口井。选择两个站点的成像在每个孔中。 </li><li>选择正确的光的波长的成像。 注:两个不同的波长（相衬和GFP荧光）被选定为延时成像。 </li><li>着眼于用激光自动对焦的井底，并采取测试图像的多个站点和不同井找到一个优化的焦平面。 </li><li>一旦焦点已经成立，开始捕获图像，每5分钟48小时为所有60口井（120点）。 </li><li>通过从HCS体系中除去96孔板喂细胞每隔24小时。从每个除去75微升培养基井，并添加100微升新鲜培养基到每个孔中（平衡在37℃和5％的CO此新鲜培养基2）。 </li><li>在时间推移实验结束时，取出96孔板从HCS系统。在无菌条件下，收集从各条件培养基样品井并且这些样品转移到另一96孔板中。 注意:这些样品可用于进一步analys是通过执行ELISA的神经营养因子。 </li><li>制备的96孔板用培养的MSC的额外分析如Ki67的细胞增殖测定或碘化丙啶活/死染色法（见下文）。 </li><li>如在下面第5节中描述为细胞迁移/细胞跟踪执行延时成像分析。 </li></ol></li></ol> 3，Ki67的细胞增殖和碘化丙啶活/死测定<ol><li> Ki67的细胞增殖试验（免疫细胞） <ol><li>冲洗的细胞培养物用0.1M磷酸盐（PO 4）缓冲液一分钟两次。固定培养，用4％多聚甲醛（PFA）进行20分钟，在室温下。取出PFA和冲洗井PBS七分钟三次。 </li><li>在最后冲洗后，加入100微升阻断溶液（磷酸盐缓冲盐水，5％正常驴血清，0.4％牛血清白蛋白，和0.2％的Triton X-100），以每孔一个次在室温下孵育1小时。 200:通过以1:1的加工率稀释于阻断溶液制备第一抗体，兔抗Ki67。 </li><li>除去阻断溶液并应用100微升的初级抗体溶液，每孔。覆盖96孔板，孵育的样品在4℃下过夜。 </li><li>次日，除去抗体溶液和冲洗用PBS为7分钟，3次。 </li><li>制备第二抗体，驴抗兔Cy3的在封闭液以1:1的加工率:500。添加的DAPI核染色的二级抗体溶液的稀释度为1:100。在除去最后的PBS冲洗后，将100μl的第二抗体/ DAPI溶液的各孔。在黑暗中在室温下孵育90分钟。 </li><li>除去二级抗体/ DAPI溶液和7分钟，3次，用PBS冲洗井每个。覆盖96孔板，并储存在4℃直至成像。 </li></ol></li><li> Propidium碘化物活/死测定<ol><li>如下所述测量细胞死亡通过碘化丙啶（PI）排除测定。 </li><li>准备以1.5μM的在培养基中的浓度的碘化丙啶染色溶液。 </li><li>加入100微升70％乙醇的MSC的一个阱2分钟以杀死这些细胞的意图。这口井用作阳性对照为PI染色。大多数干细胞将是PI染色表明细胞死亡。除去乙醇溶液。 </li><li>加入100微升的碘化丙啶染色溶液至每孔，并在5％CO 2培养箱孵育20分钟，在37℃。 </li><li>冲洗细胞，用0.1M磷酸缓冲液，1分钟，2次。固定的细胞用4％PFA中的0.1M PO 4缓冲液中20分钟，在室温下。除去PFA和冲洗用PBS三次7分钟。 </li><li>孵育用DAPI溶液（1:50）稀释于阻断溶液中1小时，在室温下进行。冲洗所有井PBS中7分钟，3次。 </li><li>取出的DAPI溶液和7分钟，3次，用PBS冲洗井每个。覆盖96孔板，并储存在4℃直至成像。 </li></ol></li></ol> 4.自动成像和多波长分析评分<ol><li>加载一个96孔板（先前处理对于Ki67的免疫标记或碘化丙啶染色的）插入HCS系统，并允许所述板中平衡20分钟，打开的HCS系统的图像采集和分析软件。 </li><li>选择用于使用照相机像素合并为1和2的增益设置查找的Z平面，其中所述细胞位于通过利用​​自动曝光功能并计算偏移为每个感兴趣的波长的10X物镜采集设置。对于这种分析，捕捉图像为DAPI（W1），Cy3标记（W2），和FITC（W3）。选择的最大强度电平，在该阴性对照孔显示为图像采集没有信号。确认此设置适合的POS可持续的竞争井。 </li><li>收购板块。捕捉图像并将其存储在图像采集和分析软件的数据库中。 </li><li>一旦图像已被收购，收购上述从图像中打开的图像采集和分析软件的离线和审查板的数据。 </li><li>选择多波长得分分析。配置的最小和最大强度为每个波长。 注:DAPI检测应当标注各地的每个可见细胞核染色。 Cy3的应检测的阳性细胞与Ki67免疫反应（IR）和下的阴性对照不应该检测红外线。对于Cy3的Ki67的红外光谱，近似最小宽度是7μm，近似最大宽度为30μm，上述局部背景的强度为150灰度级，最小染色面积为50μm2。 </li><li>所有职位运行分析。导出数据，查看电子表格。 </li></ol> 5.细胞跟踪<ol><li>打开图像习得n和分析程序，然后点击"评论板块数据[DB] ..."，选择感兴趣的板块。 </li><li>查看数据为"时间与井"。 </li><li>选择所需的选择"网站"由左击节的地点之一。选择"传送..."下的"波长"。右键单击目标以及96孔板模板，并点击"载入图像"。 </li><li>通过点击"应用程序"，然后在"跟踪对象"追踪细胞。 </li><li>使用"动态数据交换（DDE）"选择格式导出的数据， 例如，Microsoft Excel中。 </li><li>选择跟踪数据出口， 例如，经过的时间，对象，数量，距离，时间间隔，速度，绝对角度，距离的起源，增量X和增量Y。 </li><li>标签与靶细胞"Ctrl键+鼠标左键单击"利息每一个细胞。 </li><li>追踪细胞。如果有必要，停止/更改不当追踪细胞与"Esc"键，然后调整设置。 </li><li>经过细胞跟踪完成后，保存的数据与"日志数据"。 </li></ol>

<ol>
	<li>Thoenen, H., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins:+from+enthusiastic+expectations+through+sobering+experiences+to+rational+therapeutic+approaches.">Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches.</a> Nature neuroscience. 5 Suppl, 1046-1050 (2002).</li><li>Park, H., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+regulation+of+neural+circuit+development+and+function.">Neurotrophin regulation of neural circuit development and function.</a> Nature reviews. 14, 7-23 (2013).</li><li>Lin, L. F., Doherty, D. H., Lile, J. D., Bektesh, S., Collins, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GDNF:+a+glial+cell+line-derived+neurotrophic+factor+for+midbrain+dopaminergic+neurons.">GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.</a> Science (New York, N.Y.). 260, 1130-1132 (1993).</li><li>Azizi, S. A., Stokes, D., Augelli, B. J., DiGirolamo, C., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+and+migration+of+human+bone+marrow+stromal+cells+implanted+in+the+brains+of+albino+rats--similarities+to+astrocyte+grafts.">Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 3908-3913 (1998).</li><li>Prockop, D. J., Gregory, C. A., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+strategy+for+cell+and+gene+therapy:+harnessing+the+power+of+adult+stem+cells+to+repair+tissues.">One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 Suppl 1, 11917-11923 (2003).</li><li>Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+of+the+spinal+cord+following+intravenous+delivery+of+bone+marrow+cells.">Remyelination of the spinal cord following intravenous delivery of bone marrow cells.</a> Glia. 39, 229-236 (2002).</li><li>Hofstetter, C. P., 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=Marrow+stromal+cells+form+guiding+strands+in+the+injured+spinal+cord+and+promote+recovery.">Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 2199-2204 (2002).</li><li>Chopp, M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+neural+injury+with+marrow+stromal+cells.">Treatment of neural injury with marrow stromal cells.</a> Lancet neurology. 1, 92-100 (2002).</li><li>Li, 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=Transplantation+of+marrow+stromal+cells+restores+cerebral+blood+flow+and+reduces+cerebral+atrophy+in+rats+with+traumatic+brain+injury:+in+vivo+MRI+study.">Transplantation of marrow stromal cells restores cerebral blood flow and reduces cerebral atrophy in rats with traumatic brain injury: in vivo MRI study.</a> Journal of neurotrauma. 28, 535-545 (2011).</li><li>Jin, H. K., Carter, J. E., Huntley, G. W., Schuchman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+transplantation+of+mesenchymal+stem+cells+into+acid+sphingomyelinase-deficient+mice+delays+the+onset+of+neurological+abnormalities+and+extends+their+life+span.">Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span.</a> The Journal of clinical investigation. 109, 1183-1191 (2002).</li><li>Harper, M. M., 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=Transplantation+of+BDNF-secreting+mesenchymal+stem+cells+provides+neuroprotection+in+chronically+hypertensive+rat+eyes.">Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes.</a> Investigative ophthalmology &amp; visual science. 52, 4506-4515 (2011).</li><li>Arnhold, S., 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=Adenovirally+transduced+bone+marrow+stromal+cells+differentiate+into+pigment+epithelial+cells+and+induce+rescue+effects+in+RCS+rats.">Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats.</a> Investigative ophthalmology &amp; visual science. 47, 4121-4129 (2006).</li><li>Inoue, 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=Subretinal+transplantation+of+bone+marrow+mesenchymal+stem+cells+delays+retinal+degeneration+in+the+RCS+rat+model+of+retinal+degeneration.">Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration.</a> Experimental eye research. 85, 234-241 (2007).</li><li>Xia, X., Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+a+high-content+screening+approach+to+stem+cell+research+and+drug+discovery.">Concise review: a high-content screening approach to stem cell research and drug discovery.</a> Stem cells (Dayton, Ohio). 30, 1800-1807 (2012).</li><li>Scholzen, T., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ki-67+protein:+from+the+known+and+the+unknown.">The Ki-67 protein: from the known and the unknown.</a> Journal of cellular physiology. 182, 311-322 (2000).</li><li>Kapuscinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAPI:+a+DNA-specific+fluorescent+probe.">DAPI: a DNA-specific fluorescent probe.</a> Biotechnic &amp; histochemistry : official publication of the Biological Stain Commission. 70, 220-233 (1995).</li><li>Jiang, 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=Pluripotency+of+mesenchymal+stem+cells+derived+from+adult+marrow.">Pluripotency of mesenchymal stem cells derived from adult marrow.</a> Nature. 418, 41-49 (2002).</li><li>Woodbury, D., Schwarz, E. J., Prockop, D. J., Black, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+and+human+bone+marrow+stromal+cells+differentiate+into+neurons.">Adult rat and human bone marrow stromal cells differentiate into neurons.</a> Journal of Neuroscience Research. 61, 364-370 (2000).</li><li>Huang, B., Tabata, Y., Gao, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+therapeutic+agents+and+potential+targeted+gene+delivery+vehicle+for+brain+diseases.">Mesenchymal stem cells as therapeutic agents and potential targeted gene delivery vehicle for brain diseases.</a> Journal of controlled release : official journal of the Controlled Release Society. 162, 464-473 (2012).</li><li>Uccelli, A., Benvenuto, F., Laroni, A., Giunti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+features+of+mesenchymal+stem+cells.">Neuroprotective features of mesenchymal stem cells.</a> Best Pract Res Clin Haematol. 24, 59-64 (2011).</li><li>Forostyak, S., Jendelova, P., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mesenchymal+stromal+cells+in+spinal+cord+injury,+regenerative+medicine+and+possible+clinical+applications.">The role of mesenchymal stromal cells in spinal cord injury, regenerative medicine and possible clinical applications.</a> Biochimie. 95, 2257-2270 (2013).</li><li>Shi, 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=The+effect+of+lentivirus-mediated+TH+and+GDNF+genetic+engineering+mesenchymal+stem+cells+on+Parkinson's+disease+rat+model.">The effect of lentivirus-mediated TH and GDNF genetic engineering mesenchymal stem cells on Parkinson's disease rat model.</a> Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology. 32, 41-51 (2011).</li><li>Harper, M. M., 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=Brain-derived+neurotrophic+factor+released+from+engineered+mesenchymal+stem+cells+attenuates+glutamate-+and+hydrogen+peroxide-mediated+death+of+staurosporine-differentiated+RGC-5+cells.">Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.</a> Experimental eye research. 89, 538-548 (2009).</li><li>Harrill, J. A., Freudenrich, T. M., Machacek, D. W., Stice, S. L., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+neurite+outgrowth+in+human+embryonic+stem+cell-derived+hN2+cells+using+automated+high-content+image+analysis.">Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.</a> Neurotoxicology. 31, 277-290 (2010).</li></ol>

通过分子器件，LLC是支持这个视频文章出版费用。

成人间充质干细胞（MSCs）是一个有吸引力的细胞类型为一个实验策略相结合的细胞和基因递送基础的治疗的发展。的MSC是多能，能分化成中胚层谱系的细胞，并显示相当大的可塑性，分化/转分化成神经元和神经胶质细胞系用适当的感应范式17,18。此外，干细胞已被移植，并在临床前研究为许多疾病，包括神经变性条件19证明是有效的。 MSC的治疗效果是公知的，因为它们有利的抗增殖，消炎和抗凋亡活性20。的MSC也已知产生和分泌多种神经营养和生长因子，这可能有助于与移植后在损伤或疾病21的部位天真的MSC相关联的神经保护特性。 IMPOrtantly，干细胞可以被遗传修饰为持续递送的神经营养因子结合的细胞和基因治疗的应用程序，并在一些伤害或疾病的动物模型，以在CNS 11,19,22已被使用。 在开发一种组合蜂窝和基因治疗的基于策略，它是重要的细胞的健康被仔细评估之前，其在体外大量使用，特别是在体内应用。由于概念证明，我们为了研究对细胞的健康和健身的遗传修饰使用高内涵筛选（HCS）方法的后果调查设计和控制MSC线的多个群。在一般情况下，HCS是指细胞基于图像的高通量筛选14。这种筛选方法允许细胞表型的多层次空间的（细胞亚）和时间（毫秒到几天）RES的定量评估在各个实验条件olution。使用我们访问以下参数在衬底偏好可能的差异这种方法:细胞增殖，绿色荧光蛋白（GFP）的表达，细胞死亡，和细胞运动性/迁移。实验设计在96孔细胞培养板的格式。在一个单一的盘我们定期调查相对于可能的衬底相关的差异，以每个参数对慢病毒转导的细胞的不同的MSC种群，并比较了工程化的MSC与原始的，非转导的MSCs。这提供了一种对结果进行不同的MSC亚型与电池的体外测定如细胞增殖用Ki67的免疫标记，活/死细胞生存力测定法使用碘化丙啶染色的，及细胞行为通过执行时间推移数字成像直接比较装置。因为这HCS 1也可以对从个别瓦特收集条件培养基样品进行的ELISA的延伸ELLS定量测定神经营养因子的分泌。也可以使用在体外生物测定中，从不同的MSC亚型条件培养基以确定分泌因子11,23的生物活性。也可用于在体外测量从初级神经元培养物和神经干细胞系24神经突向外生长的这种类型的HCS平台。总的来说，我们的结果表明，经遗传修饰的MSC的亚群相比于未修饰的MSCs显示相似的行为。对细胞生长和细胞迁移多样培养基材的影响不是培养基材在MSC亚型之间显着不同，以及。因此，所测试的细胞外基质底物并未以在调节细胞行为这些方面对这些不同的MSC工程发挥关键作用。 这项研究表明使用的HCS系统的分析differen吨方面的细胞行为。然而，这种情况并不少见遇到与图象分析相关的局限性。上之际，同时分析该荧光图像，这是有些困难，以确定正确的阈值免疫标记或染色的细胞将被算作正标记/染色高于该。因此，为了最大限度地减少主观偏见，阈值的确定是取决于与对照（用于荧光成像阴性对照平行进行的所有处理过程中由遗漏的一次或二次抗体的）的比较。细胞迁移的使用时间推移数码影像的分析过程中的另一个限制是遇到。在一些情况下，成像软件无法小区与小区实际上迁移只有很短距离的随机布朗运动之间进行区分。额外的限制是在情况明显，其中分析软件无法区分MULTIP的存在狼疮细胞非常接近一个另一个。于分析，而不是一个完全自动化的分析过程中克服此需要限制手动小区选择。细胞铺板密度也可以导致细胞表现出更大的偏好中团块生长对细胞彼此生长在隔离种群间歪斜细胞迁移的数据。这些类型的区别是在部分可能细胞 - 基底与细胞 - 细胞偏好的反映。 使用HCS系统获取的图像，并执行数据分析提供了一个有效和迅速的方式来评估多个小区的参数。此外，常规地获得为周期使用环境室中，同时，从数小时到数天（48小时）的时间推移的数字视频用于30个不同的条件下（6底物和5种不同的MSC亚型）。这个数据随后被用于计算并确定在各个细胞系的不同的ECM中的细胞迁移率的差异分子。 在这份报告中，我们强调的高内涵筛选平台，实施，评估细胞的健康和功能。这种类型的分析是一种用于开发设计的细胞类型，以及聚合物基底，以促进定向细胞生长和神经再生合理的策略是有用的。这是朝 ​​着应用的治疗因子的干细胞系递送之前广泛的必要步骤中使用细胞移植策略体内临床前研究。

与实施有效的疗法治疗神经系统疾病的一个主要问题是开发能防止进一步恶化，也有利于功能恢复的有效方法。创新战略是基因工程干细胞体外 ，对生产要素的神经保护，他们的移植之前。该组合的基于细胞的疗法，加上一个类型的基因治疗，提供了强大的方法，疾病或损伤引起的神经元死亡的神经系统的治疗。 神经营养因子是生长和发展中的神经元的存活，以及维护和可塑性成熟的神经元必不可少的。多项研究已经证明神经营养因子显著角色在促进在中央和周围神经系统的神经元（CNS和PNS）的初始生长和分化，它们也能刺激再生在体外和一个神经损伤1 nimal车型。脑源性神经营养因子（BDNF）高度表达在CNS和在调节神经发育，突触可塑性和修理2起着重要的作用。胶质细胞源性神经营养因子（GDNF）促进许多类型的神经元，包括多巴胺能和运动神经元3的生存期。因此，对于神经修复的重要策略是提供一种神经营养因子的外源来源的神经系统的损伤或患病区域。 多能骨髓来源的间充质干细胞（MSCs）容纳用于递送治疗性蛋白质的治疗受损或患病的神经系统的巨大潜力。 MSC的移植已引起相当大的关注在努力开发患者相容的基于细胞的疗法，因为它们具有许多优点，包括:1）相对容易分离和维护的，2）多能的能力，3）小伦理问题，4-）生存和迁移移植后和5）潜在的自体移植4,5能力。有希望的结果，已报告有在动物模型中使用的幼稚和遗传工程的MSCs用于许多不同的神经变性的条件，包括脊髓损伤6,7，中风8,9，髓鞘不足10，与视网膜变性11-13。偶联细胞移植与交付从基因工程改造的干细胞的神经营养因子是一种新颖的和重要的神经修复策略。 在开发基于细胞的治疗因子递送系统的一个重要步骤是确定工程化细胞的正常健康。因此，本研究的主要目的是评估遗传工程的成体干细胞的生长的一般参数。快速评估多个小区参数的重要途径是采用蜂窝基于图像的高通screenin克（HTS），通常被称为高含量筛选（HCS）程序14。该技术允许自动图像采集和分析，这方法是特别适合于干细胞研究的应用程序。在这个项目中，我们开发了一个分析平台，允许快速定性电池基板的喜好和细胞的功能和优化与基因工程的成体干细胞采用HCS系统。

<table><tbody><tr><td>96 well plates</td><td>Greiner Bio One</td><td>655090</td><td>96 well plates selected for use in ImageXpress</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>A9647</td><td></td></tr><tr><td>Rabbit anti-Ki67 antibody</td><td>Abcam (Cambridge, MA)</td><td>Ab16667</td><td>1:200 dilution</td></tr><tr><td>Collagen type I</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>C7661</td><td>Collagen from rat tail</td></tr><tr><td>DAPI</td><td>Invitrogen (Carlsbad, CA)</td><td>D3571</td><td></td></tr><tr><td>Donkey anti-Rabbit Cy3</td><td>Jackson Immuno Res Lab (West Grove, PA)&amp;nbsp;</td><td>711-165-152</td><td>1:500 dilution</td></tr><tr><td>ECL(Entactin-Collagen-Laminin)</td><td>Millipore/Chemicon (Temecula, CA)</td><td>08-110</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone (Logan, UT)</td><td>SH30071.03</td><td></td></tr><tr><td>Equine serum</td><td>Hyclone (Logan, UT)</td><td>SH3007403</td><td></td></tr><tr><td>Ethanol</td><td>Chemistry Store (Ames, IA)</td><td>12003510</td><td>100%, 200 proof</td></tr><tr><td>Fibronectin</td><td>Fisher Scientific (Hampton, NH)</td><td>CB-40008</td><td></td></tr><tr><td>Iscove&amp;rsquo;s Modified Dulbeccos Medium (IMDM)</td><td>Hyclone (Logan, UT)</td><td>SH30396.03</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific (Hampton, NH)</td><td>P285</td><td>For PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific (Hampton, NH)</td><td>P288</td><td>For PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>L-Glutamine</td><td>Gibco/Invitrogen (Grand Island, NY)</td><td>25030-081</td><td></td></tr><tr><td>Laminine (mouse)</td><td>Trevigen (Gaithersburg, MD)</td><td>3400-010-01</td><td></td></tr><tr><td>Mouse MSCs of adult C57BL/6 mice</td><td>Tulane Cent for Gene Therapy (New Orleans, LA)</td><td></td><td>Isolated from bone marrow</td></tr><tr><td>Genetically modified MSCs (GFP, BDNF, GDNF, BDNF/GDNF)</td><td>These cells were obtained from our previous study : Ye et. al. (in preparation)</td><td></td><td></td></tr><tr><td>Normal donkey serum (NDS)</td><td>Jackson Immuno Res Lab (West Grove, PA)&amp;nbsp;</td><td>017-000-121</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Fisher Scientific (Hampton, NH)</td><td>O4042</td><td>4% PFA in 0.1 M PO&lt;sub&gt;4&lt;/sub&gt; buffer</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P0781</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P4417</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>P4707</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Invitrogen (Carlsbad, CA)</td><td>P1304MP</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>X100</td><td></td></tr><tr><td>Trypsin 0.05% (EDTA 1x)</td><td>Invitrogen (Carlsbad, CA)</td><td>25300-054</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium</td><td>Invitrogen (Carlsbad, CA)</td><td>12440&amp;ndash;046</td><td></td></tr><tr><td>Hybridoma-qualified FBS</td><td>Hyclone (Logan, UT)</td><td>SH30396.03</td><td></td></tr><tr><td>Equine serum</td><td>Hyclone (Logan, UT)</td><td>SH3007403</td><td></td></tr><tr><td>ImageXpress Micro</td><td>Molecular devices (Sunnyvale, CA)</td><td>ImageXpress micro</td><td>High content screening system</td></tr><tr><td>MetaXpress 4.0</td><td>Molecular devices (Sunnyvale, CA)</td><td>MetaXpress 4.0</td><td>Image acquisition and analysis software</td></tr></tbody></table>

high throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

MSC生长参数被培养在不同的衬底MSC的不同人群检查。 MSC亚型（干细胞，GFP-的MSC，BDNF-GFP-的MSC，GDNF-GFP-MSC和BDNF / GDNF-GFP-的MSC）的五个不同的群体接种到96孔组织培养板中预先涂有不同基板， 如图1。四天后培养，将板固定和免疫标记和/或染色的具有适当的试剂，然后检查使用HCS系统和分析与图像采集和分析软件程序进行的。 <img alt="图1" src="/files/ftp_upload/52242/52242fig1highres.jpg" width="650" /> 图1:96孔板模板为实验设计的96孔板用各种基材和孔接种工程干细胞作为所示的模板中。作为一个例子，仅WEL行高炉LS在这个实验中使用。行A，G和H留空。缩略语 - 干细胞:骨髓间充质干细胞; GFP-干细胞:绿色荧光蛋白表达干细胞; BDNF-GFP-干细胞:脑源性神经营养因子表达GFP的干细胞; GDNF-GFP-干细胞;胶质细胞源性神经营养因子表达GFP的MSCs; BDNF / GDNF-GFP-干细胞; BDNF和GDNF- GFP表达干细胞; ECL:巢蛋白，胶原蛋白IV-层粘连蛋白）。 抗Ki67免疫标记，随后用DAPI复染，被用来评估不同的基板是否影响工程化的MSC（ 图2A）的不同群体的增殖。所述的Ki67抗原的表达优先发生在细胞周期的后期G 1，S，G 2和M期，并在细胞中的休止期（G 0）未检测到，因此，是作为用于增殖15的蜂窝标记有用。 4&#39;，6-二脒基-2-苯基吲哚（DAPI）是一种常用的NU明确和染色体染液的结合后AT DNA 16的地区发出蓝色荧光。细胞在一个场的总数目可以通过计算DAPI染色的核的数目来确定。 如图2B所示 ，虽然没有在增殖MSCs的百分比变化，所有基材仍然支持大量的细胞增殖为每个MSC亚型。 <img alt="图2" src="/files/ftp_upload/52242/52242fig2highres.jpg" width="700" /> 图2:Ki67的细胞增殖试验。 （A）中合并，Ki67的免疫标记（红色）和DAPI（蓝色）染色的核的双荧光图像。许多MSC的人免疫标记与Ki67的抗体（红色）。比例尺= 50微米。例示的Ki67的免疫标记上生长聚苯乙烯（PS）的MSC亚型的百分比，聚-L-赖氨酸（PLL）（B）的棒图，纤连蛋白，胶原在体外 （DIV）I型，层粘连蛋白，巢蛋白或胶原IV-层粘连蛋白（ECL）基板5天。 N =表示一个实验。每个条表示从2口井的每个条件8成像网站平均汇总数据。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52242/52242fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 碘化丙啶（PI）染色来评价是否不同基材影响细胞存活（ 图3）。碘化丙啶是一种常用的红色荧光核和染色体染液。碘化丙啶是膜不通透性，通常排除在活细胞，因此是非常有用的，以检测死细胞群体中。死细胞的给定条件中的比例可以在与一般的核标签如DAPI组合以确定一个字段内的所有小区来确定。 PI阳性细胞的百分比是低的在检测的所有基材（图3）。作为阳性对照为PI试剂，含有干细胞的几个孔中孵育在70％的乙醇，已知杀死大多数细胞，产生的PI标记的细胞的高百分比， 如图3B和图3C的状态（乙醇处理正控制）。 <img alt="图3" src="/files/ftp_upload/52242/52242fig3highres.jpg" width="700" /> 图3:碘化丙锭的细胞死亡测定。 （A）中合并，对于碘化丙啶（红色）和DAPI（蓝色）染色的双荧光图像。虽然所有的活细胞的细胞核用DAPI染色（蓝色），无碘化丙啶染色，在干细胞。（B）中 ，几乎所有的MSCs进行染色用碘化丙啶在暴露于70％的乙醇进行检测。和比例尺在A B = 100微米。说明碘化丙啶（PI）的比例（C）条形图染色MSC亚型家养上聚苯乙烯（PS），聚-L-赖氨酸（PLL），纤连蛋白，I型胶原，层粘连蛋白，巢蛋白或胶原IV-层粘连蛋白（ECL）基板5天的体外 （DIV）。乙醇控制:这种情况曾作为阳性对照PI染色试剂。经过PI染色下面乙醇处理大部分细胞死亡，从而为PI试剂染色阳性。 N =表示一个实验。每个条表示从2口井的每个条件8成像网站平均汇总数据。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52242/52242fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 调查不同的基板上工程改造的MSCs，细胞迁移，使用时间推移数字显微镜和透射光/环境室中的HCS系统对系统进行分析的行为可能产生的影响（见补充视频1）。多个网站/孔延时成像并用于计算细胞迁移率的MSC上使用图像采集和分析软件程序的不同的衬底上生长的不同亚群。在一般情况下， 如图4C所示 ，MSC的所有亚型表现出的胞外基质包被的表面（纤连蛋白，胶原蛋白，层粘连蛋白和ECL）和最慢的未涂覆的聚苯乙烯表面上最快的迁移率。 <img alt="图4" src="/files/ftp_upload/52242/52242fig4highres.jpg" width="700" /> 图4:细胞追踪和迁移的MSCs跟踪与图像采集和分析软件。透射光和荧光图像从时间推移成像和（B）（A）开始的29小时后的时间推移成像会议结束重叠图像（见补充视频1）。细胞迁移轨迹是由彩色李肇星表示网元。比例尺:50μm的（C）的棒图表示的平均迁移率（表示为微米/小时），进行MSC亚型上生长聚苯乙烯（PS），聚-L-赖氨酸（PLL），纤连蛋白，I型胶原，层粘连蛋白，或巢蛋白胶原IV-层粘连蛋白（ECL）底物在体外 （DIV）2天。 N =表示一个实验。每个条表示至少有10成像细胞从2口井的每个条件的平均值。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52242/52242fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 总之，这些结果提供了初步证据表明，基因工程干细胞，这些细胞亚群显示类似的增长性。这些结果提供了令人信服的证据表明慢病毒介导的间充质干这些诱导使用此筛选平台研究生长参数没有检测到显着不利影响的遗传修饰。 补充视频1.定时的数字视频。迁移路径是为两个MSC〔表示为1（绿色跟踪线）和2（蓝线跟踪）]示出。在29小时内的视频捕捉。图像捕捉每5分钟。 GFP表达干细胞的荧光图像被用于时间推移成像准备的视频。校准条= 50微米。这种类型的分析是用于研究细胞的行为，包括细胞迁移和细胞分裂是有用的。

Watch this Scientific Journal Video about High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies at JoVE.com

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

high-throughput-characterization-adult-stem-cells-engineered-for

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.8K 次观看.  Iowa State University. This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

视频: High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神经科学

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

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

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 ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

医学

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

无转基因生成血液衍生神经元细胞

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...