Name: in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas
Uploaded: 2016-06-10T15:00:00.000+00:00
Duration: 9 min 31 s
Description: 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

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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	<li>Gu, G., Brown, J. R., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+tracing+reveals+the+ontogeny+of+pancreatic+cell+fates+during+mouse+embryogenesis.">Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis.</a> Mech Dev. 120, 35-43 (2003).</li><li>Kopp, J. 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=Sox9++ductal+cells+are+multipotent+progenitors+throughout+development+but+do+not+produce+new+endocrine+cells+in+the+normal+or+injured+adult+pancreas.">Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.</a> Development. 138, 653-665 (2011).</li><li>DiGiusto, D. 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=RNA-based+gene+therapy+for+HIV+with+lentiviral+vector-modified+CD34(+)+cells+in+patients+undergoing+transplantation+for+AIDS-related+lymphoma.">RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.</a> Science translational medicine. 2, 36-43 (2010).</li><li>Inada, 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=Carbonic+anhydrase+II-positive+pancreatic+cells+are+progenitors+for+both+endocrine+and+exocrine+pancreas+after+birth.">Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.</a> Proc Natl Acad Sci U S A. 105, 19915-19919 (2008).</li><li>Solar, 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=Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth.">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17, 849-860 (2009).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Jin, 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=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Fu, X., 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=MicroRNA-26a+targets+ten+eleven+translocation+enzymes+and+is+regulated+during+pancreatic+cell+differentiation.">MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation.</a> Proc Natl Acad Sci U S A. 110, 17892-17897 (2013).</li><li>Ghazalli, N., 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=Postnatal+Pancreas+of+Mice+Contains+Tripotent+Progenitors+Capable+of+Giving+Rise+to+Duct,+Acinar,+and+Endocrine+Cells+In+Vitro.">Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.</a> Stem Cells Dev. , (2015).</li><li>Jin, 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=Colony-forming+progenitor+cells+in+the+postnatal+mouse+liver+and+pancreas+give+rise+to+morphologically+distinct+insulin-expressing+colonies+in+3D+cultures.">Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.</a> Rev Diabet Stud. 11, 35-50 (2014).</li><li>Jin, 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=In+Vitro+Multilineage+Differentiation+and+Self-Renewal+of+Single+Pancreatic+Colony-Forming+Cells+from+Adult+C57Bl/6+Mice.">In Vitro Multilineage Differentiation and Self-Renewal of Single Pancreatic Colony-Forming Cells from Adult C57Bl/6 Mice.</a> Stem Cells Dev. , (2014).</li><li>Huch, 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=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. 32, 2708-2721 (2013).</li><li>Greggio, C., 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+and+conversion+of+human+pancreatic+ductal+cells+into+insulin-secreting+endocrine+cells.">Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.</a> Elife. 2, e00940 (2013).</li><li>Dorrell, C., 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+organoid-initiating+cells+in+mouse+pancreas+and+liver+are+phenotypically+and+functionally+similar.">The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.</a> Stem Cell Res. 13, 275-283 (2014).</li><li>Ku, H., Yonemura, Y., Kaushansky, K., 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=Thrombopoietin,+the+ligand+for+the+Mpl+receptor,+synergizes+with+steel+factor+and+other+early+acting+cytokines+in+supporting+proliferation+of+primitive+hematopoietic+progenitors+of+mice.">Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.</a> Blood. 87, 4544-4551 (1996).</li><li>Ku, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-expressing+colonies+developed+from+murine+embryonic+stem+cell-derived+progenitors.">Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.</a> Diabetes. 56, 921-929 (2007).</li><li>Winkler, 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=A+quantitative+assay+for+insulin-expressing+colony-forming+progenitors.">A quantitative assay for insulin-expressing colony-forming progenitors.</a> J Vis Exp. , e3148 (2011).</li><li>Gong, 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=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425, 917-925 (2003).</li><li>Formeister, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+SOX9+levels+differentially+mark+stem/progenitor+populations+and+enteroendocrine+cells+of+the+small+intestine+epithelium.">Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium.</a> Am J Physiol Gastrointest Liver Physiol. 296, G1108-G1118 (2009).</li><li>Seymour, P. 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. 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=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D. 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><tbody><tr><td>Murine ECM proteins (Matrigel)</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>354230</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Laminin Hydrogel</td><td>Provided by David Tirrell (Pasadena, CA USA)</td><td></td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Methylcellulose</td><td>Shinetsu Chemical (Tokyo, Japan)</td><td></td><td>1,500 centipoise (dynamic viscosity unit equal to 15 g/cm/sec) (high viscosity)</td></tr><tr><td>Dulbecco&#039;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>50 ml Flacon Conical vial</td><td>Corning Inc. (Corning, NY, USA)</td><td>352070</td><td></td></tr><tr><td>100 mm x 20 mm 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 -20 &amp;#176;C</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 &amp;kappa; Isotype Control</td><td>Biolegend (San Diego, CA, USA)</td><td>400522</td><td></td></tr><tr><td>Rat IgG1 &amp;kappa; 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&#039;,6-Diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>3571</td><td>Stock kept at -20 &amp;#176;C</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&#039;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 -20 &amp;#176;C</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 -20 &amp;#176;C</td></tr><tr><td>Donkey serum</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>0017-000-121</td><td>Stock kept at -20 &amp;#176;C</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,&amp;nbsp; 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>40 &amp;mu;m Filter Mesh</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m 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>1 cc Syringe</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>309659</td><td></td></tr><tr><td>10 cc 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 &amp;amp; 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 -20 &amp;#176;C</td></tr><tr><td>Vascular Endothelial Growth Factor</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>293-VE</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Activin B</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>659-AB</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Extendin 4</td><td>Sigma (St. Louis, MO, USA)</td><td>E7144</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Rspondin-1</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>3474-RS</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Falcon 5 ml Polystyrene Round-Bottom Tube</td><td>Corning Inc. (Corning, NY, USA)</td><td>352054</td><td></td></tr><tr><td>PrecisionGlide Needle 18 G x1 1/2</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>305196</td><td></td></tr><tr><td>PrecisionGlide Needle 16 G x 1 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&amp;nbsp;</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.&amp;nbsp; 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&amp;nbsp;</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><tr><td>PBS/BSA</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)</td></tr><tr><td>PBS/BSA/Dnase1</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)&lt;br/&gt;
Dnase 1 (2,400 Units/ml per pancreas)</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

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

科研

遗传学

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

发育生物学

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

生物学

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

生物工程

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

体外胰腺器官从分散的小鼠胚胎祖细胞

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

摘要

探索更多视频

此视频中的章节

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1⁺后后福格内胰腺祖细胞

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

人类多能干细胞分化为产生胰岛素的胰岛簇

一个定量测定胰岛素表达细胞集落形成先觉

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

一个系统

体外人体牙浆干细胞向胰腺血统的诱导

体外胰腺器官从分散的小鼠胚胎祖细胞

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

摘要

探索更多视频

此视频中的章节

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

人类多能干细胞分化为产生胰岛素的胰岛簇

一个定量测定胰岛素表达细胞集落形成先觉

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

一个系统

体外 人体牙浆干细胞向胰腺血统的诱导

体外胰腺器官从分散的小鼠胚胎祖细胞

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1⁺后后福格内胰腺祖细胞

体外人体牙浆干细胞向胰腺血统的诱导