我们感谢布朗露西和亚历山大Spalla从流式细胞仪分析的核心希望之城协助分拣。这项工作部分由健康（NIH）全国学院授予R01DK081587和R01DK099734到HTK，并U01DK089533以ADR，并通过国家科学基金会资助NSF-DMR-1206121和加州再生医学研究所授予RB5-07398到DAT支持受支持从约瑟夫·雅各布斯J.研究所分子工程医学在加州理工学院DAT，和来自奥克斯纳德基金会和埃拉·菲茨杰拉德基金会HTK也表示感谢。 资金来源:这项工作部分由健康（NIH）全国学院授予的支持和R01DK081587向R01DK099734 HTK，并U01DK089533以ADR，并通过国家科学基金会资助NSF-DMR-1206121和加州再生医学研究所授予RB5-07398到DAT由约瑟夫·雅各布斯杂志支持研究所分子工程医学在加州理工学院DAT，和来自奥克斯纳德基金会和埃拉·菲茨杰拉德基金会HTK也表示感谢。研究本出版物中报道包含在流式细胞仪分析的核心和光学显微镜数码影像下奖号P30CA33572卫生的国家机构的美国国家癌症研究所支持的核心完成的工作。 研究赞助商:主办单位并未参与研究设计，收集，分析或数据的解释。

伦理声明:我们坚持开展研究，以确保质量和结果的完整性广泛接受的道德标准。动物实验是根据在市灵批准的机构动物护理和使用委员会的协议进行的。 1.成年鼠胰腺准备单细胞悬液注意:在现有的出版物7,11，CD-1或B6背景的小鼠使用;这两个背景产生了相似的结果。转基因小鼠线（指定的Sox9 / EGFP）与增强型绿色荧光蛋白通过Sox9的基因位点19,20驱动的，是在CD-1后台创建。 <ol><li>安乐死用气体CO 2的1-2分钟，或直至呼吸停止3-5成年小鼠。接下来，执行对每只小鼠颈椎脱位。 </li><li>解剖和准备胰腺。 注:安乐死后尽快解剖老鼠。这是很重要的，以避免由于胰腺自身消化以事后的变化。 <ol><li>使使用剪刀鼠标的腹壁的中线垂直切口和打开腹腔。查找健脾和使用镊子轻轻提起。切脾并用剪刀胰腺的脾叶之间的结缔组织。 <ol><li>重复这个做法对胰腺十二指肠和胃瓣。放置胰腺组织中含有的冷Dulbecco改良的磷酸盐缓冲液（DPBS），0.1％牛血清白蛋白（BSA），以及青霉素和链霉素冰培养皿（P / S;完成指定为PBS / BSA溶液）。 </li></ol></li><li>使用细尖镊子解剖显微镜下去掉从胰腺脂肪组织。 注意:这是对在下面的步骤中分离的胰腺细胞的健康至关重要。 <ol><li> （可选）检查绿色荧光胰腺使用488-509 nm激发，以确保荧光体视显微镜下EGFP表达。 </li></ol></li><li>在组织培养罩，冲洗组织依次三次在3座100 25mm培养皿含有10ml冷PBS / BSA的。 </li></ol></li><li>生成组织的小块。 <ol><li>转移解剖胰腺到干燥无菌培养皿以除去尽可能多的PBS / BSA作为可能的，并且将它们传送到另一干培养皿在冰上。 </li><li>通过加入2微升DNA酶I原液（1万单位[MU] / ml）的每1ml的PBS / BSA制备含有DNA酶I的PBS / BSA中。 注意:此溶液被指定的PBS / BSA / DNA酶I.请〜200毫升该溶液中的一次，这将涉及一个分类实验。 </li><li>使用弹簧剪刀2-3分钟，或直至组织为细片剁碎的胰腺。加2-3冷PBS / BSA / DNA酶I毫升在培养皿中止他们的组织片。 </li><li>转移组织块到50ml锥形管在冰上用10毫升吸管。使总体积至10ml，用PBS / BS回收尽可能多的组织尽可能经过A / DNA酶湖</li></ol></li><li>消化胰腺饮片成多为单细胞。 <ol><li>在350-450微升添加胶原酶B（100mg / ml的股票）/ 10毫升的组织片段和孵育在37℃下将组织在水浴8分钟，漩每3-4分钟。使用10毫升注射器用16个半ģ针起草并随后喷组织溶液向下在RT 50毫升管的壁。重复此七次。 注:使用足够的力量打破了细胞簇，但避免产生气泡，可能会杀死细胞。 </li><li>返回管至水浴，在37 °℃，8分钟和纷飞每隔3-4分钟混匀。注射器组织上下七次，如上所述，并将其放置在干培养皿所述组织液的样品（10微升）。观察下一个倒，相差的组织液，用10倍物镜光学显微镜。预计单个细胞和一些小型ÇELL集群存在。 </li><li>处理冰上细胞上减缓或阻止胶原酶的活性这一点。我使用P1000移液器调整在4℃下的体积，使用冷的PBS / BSA / DNA酶I.离心机将细胞在400×g离心5分钟50毫升，和重悬在5ml冷的PBS / BSA / DNA酶的。 </li></ol></li><li>过滤器的收益率单细胞悬浮液和清洗。 <ol><li>通过一个70微米尼龙网过滤器杯，然后通过40微米尼龙网过滤器杯过滤细胞。 </li><li>悬浮细胞在5毫升冷PBS / BSA / DNA酶我和计数细胞。 注:对于2-4个月大的B6或CD-1小鼠中，每个胰腺可能产生〜5或10万个细胞，分别。这些数字可能不同实验者而异。如果过度的细胞碎片在计数发现，使体积至50ml用冷PBS / BSA / DNA酶I和离心将细胞在400×g离心5分钟，4℃。 </li><li>调整细胞悬浮液的体积，以2×10的浓度使用冷PBS / BSA / DNA酶一7细胞/ ml </li></ol></li></ol> 2.排序细胞对胰腺癌丰富菌落形成先觉注:从B6小鼠，CD133 +而不是CD133 -细胞富集PCFUs 7,9,11。 CD133 +细胞代表总数的〜13％分离的胰腺细胞被应用于11毕竟门参数。从CD-1小鼠，CD133 + Sox9的-EGFP +细胞通常代表总的胰腺细胞的〜4％，和该细胞群体富集PCFUs 7。在CD133 + Sox9的/ EGFP +细胞分选这里描述。 <ol><li>染色游离胰腺β细胞。 <ol><li>拦截整个胰腺单细胞悬液通过温育用抗小鼠CD16 / 32的单细胞悬浮液以10微克/毫升的最终浓度在冰上5分钟，以减少非特异性结合。 </li><li>除去1×10 6个细胞的等分试样（50＆＃181;升）与在5微克/毫升的最终浓度的同种型对照抗体，生物素缀合的大鼠IgG1染色，20分钟在冰上，漩每5-7分钟。 </li><li>除去1×10 6个细胞（50微升）的等分试样，并保持在冰上作为未染色的对照。 </li><li>染色细胞的其余部分用生物素缀合的抗小鼠CD133的第一抗体，以5微克/毫升的最终浓度，20分钟在冰上，漩每5-7分钟。 </li></ol></li><li>洗涤细胞。 <ol><li>通过在4℃下使体积至1ml用PBS / BSA / DNA酶I和离心机在400×g离心5分钟洗同种型对照和初级抗体染色的样品。重复此清洗步骤。 </li><li>重悬在PBS / BSA / DNA酶I同种型对照和初级抗体染色的样品，因此最终浓度为2×10 7个细胞/ ml。 </li></ol></li><li>在2＆＃染色同型控制和初级抗体染色样品链霉亲（STV）标记的别藻蓝蛋白（APC）181克/毫升的最终浓度。孵育15分钟在冰上，漩每5-7分钟。 </li><li>洗涤细胞和4&#39;，6-二脒基-2-苯基吲哚（DAPI）染色。 <ol><li>用PBS / BSA / DNA酶I洗同种型对照和初级抗体染色的样品的两倍，如在步骤2.2。重悬在PBS / BSA / DNA酶I将细胞用DAPI（0.2微克/毫升的最终浓度）。同种型对照样品的最终体积应为0.5毫升 注:确保主抗体染色样品的最终密度是适合使用的分拣机。 5×10 6个细胞的浓度/ ml的常规用于排序。 </li><li>调整未染色细胞的0.5毫升的PBS / BSA / PS / DNA酶I.体积排序前过滤通过20微米网状所有细胞，并保持细胞在冰上在暗处。 </li></ol></li><li>细胞分选。 <ol><li>使用80微米或更大的喷嘴进行排序。 注:鼠胰腺内分泌细胞很容易出现生理上的压力。何wever，鼠PCFUs不出现而引起的穿过分类器7的应力的影响。 </li><li>获取分拣机上的细胞事件和分析参数。门的细胞前向和侧向散射区域，以排除细胞碎片7。门正向和侧向散射宽度以排除细胞双峰。门排除DAPI +死细胞7。选择选通根据来自同种型的对照细胞的值（ 图1）的绿色荧光蛋白和CD133-APC信号参数。 </li><li>收集分选的细胞成含有1.5的10ml DMEM / F12培养基补充有5％胎牛血清（FCS）的5毫升聚苯乙烯管。离心机在400 xg离心排序种群为任一的DMEM / F12或PBS / BSA / DNA酶的在小体积5分钟，重悬（〜200微升）我补充有5％FCS中。使细胞保持在冰上直到使用。 </li></ol></li><li>算从分拣机获得的CD133 + Sox9的-EGFP +细胞的数目。以一个10微升细胞样品和计数细胞用血细胞计数器测定细胞密度。 推荐使用血球计，因为从分拣机机计数往往不可靠:注意。 </li></ol> 3.板块的排序细胞进入细胞集落培养含鼠细胞外基质蛋白注:请参照详细的协议单元的电镀进鼠含ECM-殖民地法在另一朱庇特公布18。 <ol><li>准备培养基。 <ol><li>制备含有DMEM / F12培养基，1％（重量/体积）的甲基纤维素，5％（V / V）的鼠细胞外基质的蛋白质，50％的培养基（体积/体积）从MESC衍生胰腺细胞样细胞的条件培养基，5％ （v / v）FCS，10毫摩尔/升烟酰胺，10纳克/ ml人重组激活素B，0.1 nmol / L的艾塞那肽-4，和1ng / ml的血管内皮生长因子-A。用于产生条件培养基的方法是18别处详细描述。 </li><li> 750毫微克/毫升RSPO-1添加到该介质如果密集落形成是期望的。 </li><li>孵育鼠细胞外基质蛋白质培养在37℃和5％CO 2的3周。 </li></ol></li></ol> 4.文化在1细胞每孔的排序细胞注:以下步骤适用于使用手和嘴移液器的单细胞操纵。另一种方法是购买一个显微。一个典型的显微包括用操纵杆操作，机动平台，倒置显微镜。 <ol><li>准备玻璃巴斯德移液器。 <ol><li>使用本生灯，绘制出巴斯德玻璃吸管的尖端细点（〜30微米的开口）。玻璃巴斯德移液管应具有棉塞以创建一个屏障，从操作者的气流。 </li><li>高压釜在121℃的火烧玻璃巴斯德移液管进行20分钟的灭菌。 </li></ol></li><li>准备96孔板文化。 <ol><li>准备冷半固体培养基一致ING到先前描述的方案18。 </li><li>分配所述介质进入以100μl的低结合平底96孔板的内孔/孔用1ml注射器。填充外井用无菌水，以保持湿度。 </li><li>广场上冰的培养皿中，直到使用。 </li></ol></li><li>准备排序细胞。 <ol><li>添加〜从分拣机收集到含10％FCS和1％甲基纤维素的5毫升聚苯乙烯管的DMEM / F12 / PS 2.5 ml终体积6000的细胞。剧烈摇晃的​​成分混合。等待5分钟或直到气泡浮到管的顶端。这一步骤并不需要在冰上进行。 </li><li>用1ml注射器用18个半ģ针分配所述细胞溶液到两张35 mm培养皿1毫升/皿。 </li><li>轻摇手工的菜流传于盘中的电池解决方案。不允许半固体培养基中在井卡恩的余量达到35mm培养皿的边缘，因为单个细胞OT明确使用倒置显微镜进行观察。 </li></ol></li><li>准备工作区围绕一个显微镜。 <ol><li>使含有DMEM / F-12培养基和1％甲基纤维素而没有任何细胞的溶液（&#39;无细胞&#39;中），并分配所述溶液（每皿1毫升）（如在4.3.2中描述）到两张35毫米的培养皿中。 </li><li>清洁和擦拭周围用70％的酒精溶液倒置相差显微镜的工作区。 </li></ol></li><li>组装与嘴部的移液器。 <ol><li>挑选在步骤4.1准备的无菌玻璃巴斯德吸管。 1毫升塑料枪头的大端附着到玻璃巴斯德吸管的顶端。 1毫升的塑料枪头的小端连接到一个薄壁的橡胶管的一端，和吹口的管的另一端。使用用于拾取同一组的多个小区在同一玻璃吸管。 </li><li>制定，由口吸入，小体积（〜10至50微升）"无细胞"半固体溶液在步骤4.4.1与玻璃巴斯德吸管制成。 注意:在玻璃巴斯德移液管的窄开口的半固体溶液产生流动阻力，并提供一屏障，以防止被拾起细胞的污染。 </li></ol></li><li>选择一个单细胞。 <ol><li>将包含在显微镜舞台上的排序细胞35毫米盘并取下盖子。 </li><li>发现用10X物镜和聚焦在合适细胞的细胞中挑选出来的。 </li><li>发现和枪头的开口放置感兴趣的细胞旁。通过口施加吸力。 注:细胞的运动应该是相当缓慢的，所以不存在的过程以后过程中丢失的细胞的风险。进入吸管的开口单元的运动应该是可见的。如果流移动过快，具有较窄的开口改变到吸管。 </li></ol></li><li>存款单细胞到文化嘛。 <ol><li>一旦该细胞是在吸液管，用舌头通过阻断口形件的开口以停止流动。从半固体培养基吸尖端，以确保半固体介质停止流动之前短暂停顿。 </li><li>放置吸管尖成井在步骤4.2中制备的96孔板中。轻轻吹嘴部推动细胞慢慢地。标记井的细胞被沉积后，为了避免两个小区镀入井中。 </li></ol></li><li>确保单个细胞在文化很好地。 <ol><li>将枪头在"无芯"步骤中4.4.1准备。同时观察尖端的显微镜下的开口，压出剩余的半固体溶液。期望无细胞流出吸管尖。 </li><li>仔细检查，发现在文化以及那​​里的单细胞植入的位置。 </li></ol></li><li>培养在5％的单电池在37℃ 的 CO 2达到3周。 </li></ol> 5.从半固体培养微流控定量RT-PCR分析挑选单个菌落注:三个星期后镀排序CD133 + Sox9的/ EGFP +细胞进入鼠含有ECM集落测定法中，环或密集菌落形成7（ 图2）。当振铃/密菌落离解成单细胞悬浮液，并重新接种到层粘连蛋白凝胶培养，被后产生内分泌/腺泡菌落大约一周7（ 图2）。确定每个集落的细胞系组成，微流体定量RT-PCR分析被用于检测系标记7的表达。对于预扩增，集落是在含有TaqMan探针混合，反应缓冲液，和的SuperScript III 21主混合物混合。的48.48阵列芯片随后用于微流体PCR反应21。 <ol><li>制备预扩增PCR混合含有48探头。 <ol><li>吸取1.5μl，以1.5毫升管各的TaqMan探针（20倍的股票）的。添加78微升TE缓冲液中以调节音量，以150微升。 </li></ol></li><li>从制造商21以下协议准备主结构。 </li><li>将9微升混合液到每个0.2毫升薄壁反应管适合PCR和重复此步骤达48管。 </li><li>匹克从半固体培养单菌落。 注:环/密克隆比内分泌/腺泡菌落较大，直径从50〜400微米9,11。因此，单环/密菌落使用与该被弯曲成弯曲形状的10微升枪头配备一个P10移液器拾取。采摘小内分泌/腺泡集落（ 图2），请参阅步骤4。 <ol><li>找到的在高倍率下（ 例如 ，20X物镜）利益群体，缩小放大率，而aspiratË殖民地。 （可选的）取菌落的相位对比图像在此步骤以记录菌落的可见特性。 </li><li>转移菌落到含有9微升主结构的PCR管。 </li><li>选择下一个殖民地。在总额高达45殖民地政策有可能根据PCR运行进行分析，留下3点的控制。 </li></ol></li><li> RNA提取和cDNA合成用预扩增。 <ol><li>大力进行热反应前VORTEX样品。 </li><li>根据制造商的说明21执行热循环的反应。戒指/密克隆需要14个周期，内分泌/腺泡殖民地需要16-20个周期，单细胞需要22个周期。 注意:预扩增的cDNA可以存储在之前的微流体PCR分析-20℃。 </li></ol></li><li>运行随后的PCR反应中，使用微流体芯片，根据制造商的说明21。 </li></ol><p类="jove_title"&gt; 6。殖民地全山免疫染色<ol><li>嘉实和修复殖民地。 <ol><li>拿起殖民地在显微镜下，在步骤4或5.4，并将其添加到4％多聚甲醛溶液。孵育O / N在4℃轻轻摇动。 </li><li>洗净用PBS殖民地两次，在室温10-30分钟。与石蜡密封1.5毫升管存储固定殖民地在4℃。 </li></ol></li><li>染色与抗体的殖民地。 <ol><li>转移的菌落到96孔黑色板的一个孔与含有200微升封闭缓冲液（5％驴和/或山羊血清，0.1％的Triton X-100在1×PBS中）一个透明底部。孵育O / N在4℃轻轻摇动。 </li><li>稀释一级抗体对预先确定的浓度:用封闭缓冲液（ 例如 ，1 500为仓鼠抗粘蛋白1抗体）。转移菌落到一个干净的孔用200μl初级抗体，并轻轻摇动孵育O / N在4℃。 W¯¯用PBS含有0.1％Tween 20的转移的菌落到一个新的井用200μl1×PBS中的/ 0.1％吐温20 10分钟在RT每次洗涤灰分菌落3次。 </li><li>稀释二级抗体，以预先确定的浓度（ 例如 ，1:2,000山羊抗亚美尼亚仓鼠抗体）使用封闭缓冲液。保持在黑暗中的溶液。转移菌落清洁含有200微升的二级抗体的孔中，并在室温下孵育2小时。用PBS洗菌落3次/ 0.1％吐温20，如在步骤6.2.2。 </li></ol></li><li>反染色菌落DAPI和可视化与激光共聚焦显微镜。 <ol><li>转移到菌落井含300nM的DAPI PBS并在室温下孵育5分钟。 </li><li>转移DAPI染色殖民地35毫米的玻璃底培养皿可视化。广场上的殖民地，以防止水分蒸发的顶部盖玻片。 </li><li>使用共聚焦显微镜捕捉图像。为了形象化DAPI stainin克，使用730-950纳米双光子激发波长。使用氩激光以458纳米的波长来激发与519和561纳米的发射波长的荧光染料。使用一个氦氖激光器的波长633nm具有665nm的发射波长来激发荧光染料。 </li></ol></li></ol> 7.分离并重新盘主环/密集殖民地变成次要殖民地化验注意:所有程序应当在无菌条件下进行。避免冷休克的细胞在此过程中尽可能多地，例如将细胞在冰上或洗涤细胞用冷PBS / BSA中。这种做法减少再镀细胞的活力。 <ol><li>准备解决方案。 <ol><li>预温水洗涤缓冲液（DMEM / F12; P / S; 0.1％BSA）在37℃水浴中。 </li><li>预温96孔板（平底;低结合）在37℃培养箱。加入100μl100％FCS的在板的一个孔与100μl的0.25％胰蛋白酶-EDTA溶液添加到第二好。 </li></ol></li><li>收集殖民地。 <ol><li>挑选和池共使用10微升枪头原代培养物20个或更多环/密菌落，如在步骤5.4中描述。 </li><li>放置菌落在温暖（至少RT）洗涤缓冲液（〜1000微升）在1.5ml管中并旋转，在400×g离心5分钟。去除上清。 </li></ol></li><li>离解成菌落单细胞悬液使用胰蛋白酶。 <ol><li>传输剩余量（20微升或更小），它包含菌落到包含暖胰蛋白酶溶液（在7.1制备）的井。 </li><li>孵育所述板在37℃培养箱（未水浴）1.5分钟。取出板和吸管殖民地几次。在37℃下再1.5分钟。 </li><li>取出板和吸管上下几次，打破了殖民地。检查在显微镜下看能否克隆已主要分散成单细胞悬液。避免殖民地的过度消化。 </li></ol></li><li>停止添加FCS的胰蛋白酶反应。 <ol><li>转移100μl的温暖的FCS到包含细胞的孔。移液器上下几次。转移细胞到含1000微升温水洗涤缓冲液1.5 ml的管中。离心在400×g离心在RT 5分钟。 </li><li>用温缓冲液洗两次。 </li><li>重新悬浮细胞在〜200μl的缓冲液或培养基。 </li></ol></li><li>算使用血细胞计数器单元的数目。保持细胞悬浮液在RT。 </li><li>重板解离细胞到含或鼠ECM蛋白（5％体积/体积）或层粘连蛋白的水凝胶（100微克/毫升）次级菌落测定并用5％的CO 2孵育细胞在37℃。 注:对于重新电镀细胞进入小鼠细胞外基质蛋白，使用每孔和文化2,500-5,000细胞2-3周。对于重新进入pating层粘连蛋白凝胶文化，用每孔和文化10,000-25,000细胞7-12天。 </li&gt; </ol>

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A., 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=A+dosage-dependent+requirement+for+Sox9+in+pancreatic+endocrine+cell+formation.">A dosage-dependent requirement for Sox9 in pancreatic endocrine cell formation.</a> Dev Biol. 323, 19-30 (2008).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81, 2844-2853 (1993).</li><li>Suzuki, A., Nakauchi, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+multipotent+pancreatic+progenitors+using+flow-cytometric+cell+sorting.">Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.</a> Diabetes. 53, 2143-2152 (2004).</li><li>Seaberg, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+adult+pancreatic+exocrine+cells+to+beta-cells.">In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.</a> Nature. 455, 627-632 (2008).</li><li>Baeyens, 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=Transient+cytokine+treatment+induces+acinar+cell+reprogramming+and+regenerates+functional+beta+cell+mass+in+diabetic+mice.">Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.</a> Nat Biotechnol. 32, 76-83 (2014).</li><li>Thorel, F., 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=Conversion+of+adult+pancreatic+alpha-cells+to+beta-cells+after+extreme+beta-cell+loss.">Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.</a> Nature. 464, 1149-1154 (2010).</li></ol>

作者什么都没有透露。

胰腺菌落测定和单个菌落分析这里描述通过在解密造血祖细胞的生物学在过去几十年23都发挥主要作用的含有甲基纤维素造血集落测定法启发。在这些测定法（ 图5），解离胰腺细胞铺到与支持环，密集或内分泌/腺泡菌落7的形成适当的生长因子和ECM蛋白半固体培养基含有甲基纤维素。那就是能产生细胞的殖民地单祖细胞被称为PCFU。通过表征采用实时定量微-PCR和全挂?...

胰腺是由三个主要的细胞谱系;腺泡细胞分泌消化酶，导管分泌粘蛋白，抵御病原体和运输消化酶肠道，内分泌细胞分泌的激素，包括胰岛素和胰高血糖素，即保持血糖平衡。在胰腺的胚胎发育，早期导管细胞能够在成年动物1,2的胰腺引起三个谱系三效祖细胞的来源。因为成人干细胞和祖细胞，如骨髓干细胞，已成功地用于治疗各种疾病3，有在成人胰腺找到干细胞和祖细胞的强烈的兴趣。如果隔离和成人胰腺干细胞和祖细胞的操纵是可能的，这些细胞可用于治疗疾病，例如1型糖尿病，其中所述胰岛素分泌细胞是由自身免疫破坏。 使用体内 CRE-LOX谱系追踪技术，稻田和同事表明，标记有标记物，碳酸酐酶II成年鼠导管细胞，会引起所有三个胰腺谱系4。然而，使用其他导管标记物，如HNF1B 5和Sox9的2，可以得出结论，导管细胞不是在成年小鼠的β细胞的主要来源。 几年前，提出的是，上述讨论的原因，可能是由于缺乏，在字段6,7，的可以用于测量的自我更新和多谱系分化两个标准需要适当的分析工具定义的干细胞。提到体内 CRE-LOX谱系追踪技术以上可为在群体水平的祖后代关系的证据。然而，这一谱系追踪技术在其权力仅限于辨别是否单系祖细胞可以自我更新和分化成多种谱系。因为如果几个单效祖细胞，每一个不同的分化潜能，一起分析，它们可共同出现有多谱系分化能力的单细胞分析是重要的。此外，干细胞是通常成人器官的次要人口。一个次要的细胞群的活动可以由主要的人口被屏蔽。因此，从人群研究的否定结​​果并不一定表示不存在的干细胞。最后，CRE-LOX谱系追踪目前不允许自我更新的测量。 开始解决在胰腺祖细胞生物学，细胞集落领域的技术间隙7-11或类器官12-15 </suP&gt;使用三维培养系统测定法设计。胰腺祖细胞的两个集落测定法在我们实验室开发了:一个含有鼠细胞外基质蛋白（ECM）（见方法和设备表 ）的市售制剂，以及另一个包含层粘连蛋白的水凝胶，限定人造的ECM蛋白7-11。祖细胞在含有半固体培养基的甲基纤维素进行混合。甲基纤维素是从木纤维制得的生物惰性和粘性材料，并已在造血细胞集落测定法16被经常使用。半固体培养基中含有甲基纤维素，限制了单一祖细胞的运动，使得它们不能重新聚集。然而，该介质是足够软以允许祖细胞生长和分化成在三维空间中的细胞的集落。继血液学家的传统，这是能够引起细胞集落胰腺祖细胞为:NAmed的胰腺细胞集落形成单位（PCFU）。 PCFUs，在小鼠含ECM-集落培养生长时，引起了被命名为"环"殖民地7囊性殖民地。在添加的Wnt激动剂，R-spondin1，成鼠含ECM-文化，一些环殖民地变成"密"殖民地7。在本文中，这两种类型的小鼠的ECM培养中生长的菌落被统称为"环/密"菌落。当戒指/高密度菌落分离成单细胞悬液，重新接种到含有层粘连蛋白凝胶文化，"内分泌/腺泡"菌落形成7。 使用单个菌落分析，发现大多数环/密集和内分泌/腺泡殖民地，无论是从成人（2-4个月大）7,11或青年（1周龄）9鼠胰腺，表达所有三个系标记。这表明大多数始发PCFUs的是三效。在小鼠含ECM-集落培养，成年鼠PCFUs有力地自我更新和扩大了11周约50万次文化7。鼠的ECM优先支持导管细胞过度内分泌和腺泡谱系的分化，而 ​​在层粘连蛋白的水凝胶的情况下，被鼓励鼠PCFUs优先分化成内分泌和腺泡细胞和少这样的导管谱系7,9,11。重要的是，胰岛素+胰高血糖素-单-激素细胞在层粘连蛋白凝胶培养产生并且响应分泌胰岛素对葡萄糖刺激体外 7,9，提示功能成熟。三谱系分化潜能7,9和个人PCFUs的自我更新11由单细胞显微操作， 即证实，培养每孔一个细胞集落形成。总之，这些结果提供的证据表明有自我更新，三正POTENT，祖细胞样细胞在出生后小鼠胰腺，显示在三维培养的活动。 本文中描述的鼠PCFU测定是从现有集落检测设计用于从小鼠胚胎干细胞（mESCs）17分化的祖细胞。该协议是在另一个朱庇特出版物18记载的细节。培养组件和执行成人PCFUs鼠含ECM集落测定法所需的技术是一样的MESC衍生的祖细胞17,18。因此，测定的这些方面将不在这里重复;代替下列程序将得到解决:1）成人胰腺分离和分拣CD133 + Sox9的/ EGFP +导管细胞，从成年小鼠7,2）的分选的细胞的单细胞操纵，3）单菌落丰富PCFUs分析使用微流体的qRT-PCR和整体卡口免疫染色，和4）科勒尼的离解s转换单细胞悬浮液，重新电镀成鼠细胞外基质或层粘连蛋白的水凝胶菌落测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Murine ECM proteins (Matrigel)</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>354230</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Laminin Hydrogel</td>
			<td>Provided by David Tirrell (Pasadena, CA USA)</td>
			<td></td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Shinetsu Chemical (Tokyo, Japan)</td>
			<td></td>
			<td>1500 centipoise (dynamic viscosity unit equal to 15g/cm/s) (high viscosity)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Mediatech (Manassas, VA, USA)</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Flacon Conical vial</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100mmx20mm Suspension culture dish</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase1</td>
			<td>Calbiochem (Darmstadt, Germany)</td>
			<td>260913</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche (CH-4070, Basel, Schweiz, Switzerland)</td>
			<td>11088831001</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>101310</td>
			<td>low endotoxin, azide free</td>
		</tr>
		<tr>
			<td>PE-Cy7 Rat IgG2a &#954; Isotype Control</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>400522</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 &#954; Isotype Control</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-4301-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133-Biotin</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-1331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD71-PE-Cy7</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>113812</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Allophycocyanin</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>405207</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>3571</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mucin 1</td>
			<td>Thermo Fisher Scientific (Waltham, MA USA)</td>
			<td>HM-1630-P1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 649 Goat anti-Armenian Hamster</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>127-495-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Mediatech(Manassas, VA, USA)</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Tissure Culture Biologicals (Long Beach, CA, USA)</td>
			<td>101</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Tris Ethylenediaminetetraacetic acid</td>
			<td>TEKnova (Hollister, CA, USA)</td>
			<td>T0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy Micro Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTec Reverse Transcription Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>205310</td>
			<td></td>
		</tr>
		<tr>
			<td>CellsDirect One-Step qRT-PCR Kit</td>
			<td>Ambion/Invitrogen(Grand Island, NY, USA)</td>
			<td>11753-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Santa Cruz Bio (Santa Cruz, CA, USA)</td>
			<td>SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>005-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>0017-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T-4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies (Waltham, MA, USA)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15230-147</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>13-678-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>40um Filter Mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um filter mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 96 Well Suspension</td>
			<td>Sarstedt</td>
			<td>83.3924 (Previously 83.1835)</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr>
			<td>48.48 Dyanmic Array Chip</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>BMK-M-48.48</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidigm GE 48.48 Dynamic Array Sample &#38; Assay Loading Reagent Kit</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>85000800</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems (Grand Island, NY, USA)</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol sorbitan monolaurate</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dish</td>
			<td>MatTek (Ashland, MA, USA)</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm petri dish with glass bottom</td>
		</tr>
		<tr>
			<td>Mouth Piece/ Rubber Tubing</td>
			<td>Renova Life Inc. (College Park, MD, USA)</td>
			<td>MP-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>N0636</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>293-VE</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Activin B</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>659-AB</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Extendin 4</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>E7144</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Rspondin-1</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>3474-RS</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Falcon 5mL Polystyrene Round-Bottom Tube</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 18Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 16Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305198</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Ultra-Low Attachment Surface 24 well flat bottom plate&#160;</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 96 Black Well Plate</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3603</td>
			<td>Flat, clear bottom with lid.&#160; Black polystyrene TC-treated microplates</td>
		</tr>
		<tr>
			<td>Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope</td>
			<td>Carl Zeiss AG (Oberkochen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomark HD&#160;</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aria Special Order Research Product Cell Sorter</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas

茎和从成人胰腺祖细胞可以是治疗性的β样细胞为1型糖尿病治疗的患者的潜在来源。然而，它仍然是未知在成人胰腺中是否存在干细胞和祖细胞。使用CRE-LOX在成年小鼠世系追踪研究策略已经初见成效，无论是支持或反驳，β细胞可以从管道，那里的成人胰腺祖细胞可能存在于假定的位置而产生的想法。 这些体内 CRE-LOX谱系追踪方法，但是，不能回答的自我更新和多谱系分化两个必要定义一个干细胞的标准的问题。要开始解决这个技术差距，我们发明了胰腺祖细胞的三维殖民地化验。我们最初公布后不久，其他实验室自主研发的类似，但不完全相同，方法调用的类器官检测。相比于类器官测定中，我们的方法是采用甲基纤维素，其形成粘性溶液，允许在低浓度包含细胞外基质蛋白。含甲基纤维素的测定法允许容易检测和祖细胞的单细胞水平，当祖细胞构成的小亚群这是至关重要的分析，因为对于许多成人器官的干细胞的情况下。一起，从几个实验室结果表明在小鼠胰腺祖细胞样细胞的体外的自我更新和多谱系分化。目前的协议描述了两个基于甲基纤维素集落实验表征小鼠胰腺祖细胞;一个含有鼠细胞外基质蛋白的市售制剂和其它人工细胞外基质蛋白已知为层粘连蛋白水凝胶。这里示出的技术是:1）的胰腺解离和CD133 +的Sox9 / EGFP的排序从成年小鼠+导管细胞，2）在S的单细胞操作orted细胞，3）单菌落分析使用微流体的qRT-PCR和整体卡口免疫染色，和4）主菌落解离成单细胞悬液，并重新电镀到次级菌落测定以评估自我更新或分化。

成人胰腺祖细胞可以通过荧光激活细胞分选（ 图1）来富集。这里使用的Sox9的/ EGFP转基因小鼠线作为GENSAT脑图谱项目19的结果被首先产生，并且EGFP报告是含有细菌人工染色体的控制下〜75 kb的上游和〜150 kb的Sox9的20的下游序列。在这些小鼠中，EGFP高效和特异性22标签胰管。胰腺采购和离解成单细胞悬浮液，并与生物素缀合的抗CD133抗体?...

Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

体外集落测定法来检测自我更新和从成年鼠胰腺分离的祖细胞的分化被设计的。在这些测定中，胰祖细胞产生细胞集落在3维空间中含有甲基纤维素半固体培养基。用于处理单个细胞和单独的菌落的特征的协议进行了描述。

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

in-vitro-colony-assays-for-characterizing-tri-potent-progenitor-cells

Research

JoVE Journal

Developmental Biology

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

9.4K 次观看.  Beckman Research Institute of City of Hope.  体外集落测定法来检测自我更新和从成年鼠胰腺分离的祖细胞的分化被设计的。在这些测定中，胰祖细胞产生细胞集落在3维空间中含有甲基纤维素半固体培养基。用于处理单个细胞和单独的菌落的特征的协议进行了描述。 

视频: In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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 ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

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 ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

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 ...