Name: chondrogenic pellet formation from cord blood-derived induced pluripotent stem cells
Uploaded: 2017-06-19T15:00:00.000+00:00
Duration: 12 min 10 s
Description: Watch this Scientific Journal Video about Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

这项工作得到韩国卫生福利和家庭事务部韩国医疗保健技术研发项目（HI16C2177）的资助。

该协议由韩国天主教大学机构审查委员会批准（KC12TISI0861）。用于重新编程的CBMC直接从首尔圣玛丽医院的脐血库获得。 来自iPSCs的软骨形成分化<ol><li> CBMC-iPSC一代 <ol><li>使用我们以前的工作23所示的协议生成CBMC-hiPSC。 </li><li>将血细胞收集在15 mL锥形管中，并使用血细胞计数器进行计数。 </li><li>准备3×10 5个细胞，并在515×g和RT下离心5分钟。通过抽吸弃去上清液，并将细胞重新悬浮于0.5mL血细胞培养基中。 </li><li>将细胞转移到未包被的24孔板的孔中，并按照制造商的建议添加仙台病毒混合物。 </li><li>在1150 xg和30℃下将板离心30分钟。 </li><li>前前后后离心，将细胞在37℃，5％CO 2中孵育过夜（O / N）。 </li><li>第二天，将转导的细胞转移到基质涂层的孔中。在30℃下以1,150×g离心板30分钟。 </li><li>离心后，取出上清液，加入1mL iPSC培养基，并将细胞O / N在37℃下保持在5％CO 2中 。 </li><li>在37℃和5％CO 2下保持连接的细胞。每天更换培养基，用新鲜的iPSC培养基更换培养基。 注意:殖民地将在转导后第14-21天出现。 </li></ol></li><li> EB一代 <ol><li>在37℃和5％CO 2下保持hiPSC。每天更换培养基，用新鲜的Essential 8（E8）培养基代替。 </li><li>在E8培养基中的玻连蛋白包被的100毫米皿中制备2×10 6个hiPSC。 </li><li>从培养皿中取出E8培养基，用无菌磷酸盐缓冲盐水（PBS）洗涤。 </li><li>添加1mL的1mM乙二胺四乙酸（EDTA），并在37℃和5％CO 2下孵育2分钟。 </li><li>用3mL E8培养基收获细胞并将其转移到新的15-mL锥形管中。在250×g室温（RT）下将细胞离心2分钟。 </li><li>吸出上清液而不干扰细胞沉淀，并将细胞重悬于5mL E8培养基中。 </li><li>使用血细胞计数器计数细胞，并为每100 mm培养皿制备2×10 6个细胞。将制备的细胞重悬于10μME8和EB培养基（1:1）与10μMrho相关激酶（ROCK）抑制剂的混合物中。 </li><li>孵育细胞O / N用于在37℃和5％CO 2下聚集。 </li><li>在第二天，通过吸移收集聚集的EB。以250 xg离心细胞1分钟。去除上清液并将EB重悬于10mL新鲜E8培养基中。 </li><li>将生成的EB放大5天，每日更换一次h E8中等。为了进一步成熟，将培养基更换为E7培养基。将EB保持在37℃和5％CO 2另外5天，用新鲜E7培养基进行每日中等变化。 注意:E7培养基是不含FGF2的E8培养基。 </li></ol></li><li> 来自EBs的生长细胞诱导 <ol><li>加入6 mL 1％明胶至100 mm培养皿，37℃和5％CO 2孵育30分钟。 </li><li>取出明胶，然后将其完全干燥2-3小时，然后再使用。 </li><li>将EB转移到50mL锥形管中。允许EB沉降到锥形管的底部。去除上清液而不干扰EBs。 </li><li>将EB重悬于10mL含有20％胎牛血清（FBS）的Dulbecco&#39;s Modified Eagle&#39;s Medium（DMEM）中。将EB转移到明胶包被的100毫米的盘子上。加入10μMROCK抑制剂。 </li><li>孵育并保持细胞在37℃和5％CO 2 7天。改变主题每隔一天进行一次，不添加ROCK抑制剂。 </li></ol></li><li> 软骨形成颗粒形成 <ol><li>从培养皿中吸取培养基，用PBS洗涤细胞三次。 </li><li>应用1mL的1mM EDTA，并在37℃和5％CO 2下孵育2分钟。 </li><li>使用5 mL含20％FBS的DMEM收获细胞，并将其转移到新的15 mL锥形管中。 </li><li>以250×g和RT离心细胞2分钟。丢弃上清液并将沉淀重悬于10mL含有20％FBS的DMEM中。 </li><li>使用40μm的细胞过滤器过滤和丢弃细胞团，并收获单个细胞。使用血细胞计数器计数单细胞。 </li><li>以250×g和RT离心细胞2分钟。取出上清液，每粒沉淀在带有300μL软骨形成分化培养基（CDM）的15mL锥形管中种子3×10 5个细胞。 </li><li>对于沉淀物形成，将细胞离心5分钟680 xg和RT。 注意:在软骨形成颗粒的分化期间，颗粒保持在15mL锥形管中。 </li><li>在37℃和5％CO 2下孵育细胞过夜。 </li><li>每2-3天更换一次CDM。 3天内，颗粒呈现扁平的球形形态。在37℃和5％CO 2下保持颗粒21天。 </li></ol></li></ol> 2.染色体软骨细胞表征<ol><li> 软骨形成嵌入 <ol><li>在包埋之前，在58℃制备和熔融石蜡。 </li><li>将小丸固定在1mL的4％多聚甲醛中，在室温下在1.5mL管中固定2小时。 注意:多聚甲醛是剧毒的。避免接触眼睛，皮肤或粘膜。尽量减少暴露并避免吸入，同时准备。 </li><li>将一层纱布放在盒上，并使用移液管转移固定的颗粒。通过折叠覆盖颗粒请将纱布盖好并关上盒盖。 </li><li>在100ml 70％乙醇（EtOH）中开始脱水两次，每次10分钟。通过在80％和95％EtOH中连续10分钟洗涤来使沉淀物脱水。 </li><li>将颗粒转移到100％乙醇中10分钟。用新鲜的EtOH重复三次。 </li><li>对于"清除"，将溶液与EtOH和二甲苯的100mL 1:1:1混合物，然后用1:2的EtOH和二甲苯混合物交换10分钟。通过在100％二甲苯中孵育两次，每次10分钟，清除剩余的EtOH。 </li><li>对于石蜡浸润，将颗粒在连续的二甲苯和石蜡混合物中孵育。在58°C进行整个石蜡浸润过程。将颗粒在100ml二甲苯和石蜡的2:1混合物中孵育30分钟。 </li><li>将溶液换成100ml 1:1的二甲苯和石蜡混合物，并孵育30分钟。 </li><li>将溶液换成100mL的二甲苯和石蜡混合物，并孵育3次0分钟</li><li>为了最终的渗透，将颗粒转移到100％石蜡的第一浴中并孵育2小时。 </li><li>将颗粒转移到100％石蜡的第二浴中，并在58℃下孵育O / N。 </li><li>第二天，使用镊子轻轻地将颗粒转移到模具上。从石蜡分离器中将石蜡加入到模具中。在4℃下固化石蜡30分钟。 </li><li>将部分切割成7μm，并将部分转移到载玻片上。让幻灯片干燥过夜，并将载玻片存放在RT中，直到它们准备使用。 </li></ol></li><li> 幻灯片准备 <ol><li>通过将100 mL，100％，90％，80％和70％EtOH依次移动5 min，使载玻片脱蜡。 </li><li>将载玻片放在玻璃瓶中，用自来水冲洗5分钟。 </li></ol></li><li> 阿蓝染色 <ol><li>将载玻片在50mL的1％阿尔卑斯蓝溶液中孵育30分钟。 不E:将Alcian蓝在3％乙酸溶液中稀释。使用乙酸将pH调节至2.5。 </li><li>将载玻片放在玻璃瓶中，用自来水冲洗2分钟。 </li><li>用去离子水（DW）冲洗载玻片并用核快速红色溶液复染2分钟。 </li><li>将载玻片放在玻璃瓶中，用自来水冲洗1分钟。 </li><li> 2.3.5）在步骤2.6中进行脱水和安装幻灯片。 </li></ol></li><li> 甲苯胺蓝染色 <ol><li>将载玻片在50mL甲苯胺蓝溶液中孵育4分钟。 </li><li>将载玻片放在玻璃瓶中，用自来水冲洗5分钟。 </li><li>在步骤2.6中继续脱水并安装幻灯片。 </li></ol></li><li> 免疫组织化学染色 <ol><li>在3％H 2 O 2中孵育载玻片15分钟用于内源性过氧化物酶阻断。 </li><li>应用200μl在tris-bu中以1:100稀释的一抗（COL1A1和-COL2A1抗体）将含有1％牛血清白蛋白（BSA）和5％正常山羊血清（NGS）的盐水（TBS）置于载玻片上并在4℃下孵育O / N。 </li><li>第二天将载玻片置于玻璃瓶中，用含有0.1％聚山梨酯20（TBST）的TBS洗涤三次，每次5分钟。 </li><li>将200μL二抗（山羊抗兔IgG抗体，在含有1％BSA和5％NGS的TBS中1:200稀释）稀释至载玻片中并在室温下孵育40分钟。 </li><li>将载玻片放在玻璃瓶中，用50 mL洗涤3次，每次5分钟。 </li><li>对于信号扩增，应用HRP-偶联物链亲和素溶液至载玻片并孵育10分钟。 </li><li>将载玻片放在玻璃瓶中，用50 mL洗涤3次，每次5分钟。 </li><li>混合DAB-过氧化物酶底物溶液，每个载玻片应用200μL溶液，孵育1分钟。 </li><li>将载玻片放在玻璃瓶中，用自来水冲洗5分钟。 </li><li>染液用Mayer&#39;s苏木精处理1分钟。 </li><li>用DW清洗幻灯片。 </li><li>在步骤2.6中继续脱水并安装幻灯片。 </li></ol></li><li> 脱水和安装 <ol><li>通过将它们依次移动通过100mL的70％，80％，90％和100％EtOH，每次30s，使载玻片脱水。 </li><li>将载玻片浸入两个二甲苯变化中，每次1分钟。 </li><li>在盖玻片上加入50μL安装溶液，并将幻灯片放在顶部。 </li></ol></li></ol>

作者没有什么可以披露的。

该协议成功地从CBMCs生成了hiPSC。我们使用含有山中因素的仙台病毒载体24重新编程CBMCs到hiPSC。分化使用3例，所有实验均使用该方案成功生成软骨细胞团。许多研究报道了hiPSC分化为软骨细胞25,26,27,28的方案。然而，需要进一步的研究来确认使用CBMC-hiPSCs作为软骨再生和恢复的候选者。 使用由各种体细胞类型11,25,26,27,29产生的hiPSC证实软骨形成。许多报告显示重编程中使用的体细胞的起源可影响hiPSC 30,31,32,33的分化结果。脐带血是HSC和MSC丰富的来源。没有关于脐带血来源细胞如血细胞或MSC之间差异的报道。然而，以前的研究表明，从脐带血或皮肤成纤维细胞产生的hiPSCs，关节软骨细胞衍生的hiPSC更可能通过软骨形成。使用由成纤维细胞，心脏祖细胞和内皮细胞产生的hiPSC的内皮分化的结果表明，起始组织可以反映终末分化的iPSC衍生中的组织特异性体细胞记忆，特别是在早期传代之间，10和20 34之间。在hiPSCs的早期传代中，内皮细胞衍生物ved hiPSCs更有效地分化为内皮谱系。然而，这些细胞系之间的显着差异在hiPSC超过20代中消失。因此，在临床研究中使用hiPSCs时，仔细考虑早期传代中携带的体细胞记忆是非常重要的。 近来，已经开发了各种修复缺损关节软骨的方法。微骨折通过在骨中钻多个孔来诱导骨髓的自然修复35 。膝关节置换术，也称为膝关节置换术，用于替代损伤的软骨。然而，微裂缝对于严重的关节软骨损伤无效，用于膝关节置换的仪器必须每10〜15年更换一次。 这些天，基于细胞的疗法是软骨修复的有前途的替代方法。自体软骨细胞植入（ACI）较宽用于基于细胞的软骨恢复。 ACI通过将自体软骨细胞直接注入缺陷来完成。然而，自体软骨细胞在体外培养条件下变成成纤维细胞样细胞。在自体软骨细胞的收获过程中，额外的损伤也是不可避免的。已经建议MSC作为软骨恢复的细胞来源。脐血是用于工程软骨的MSC的有用且可及的细胞来源。然而，分离脐血MSC的成功率是有争议的， 37,38 。许多研究报道了脐血MSC的成功分离。几项研究表明，脐带血量是影响MSC隔离产率36,39的关键参数。为了获得高质量的MSC，细胞的通过也是至关重要的。以前的研究表明在一定细胞分裂数之后，MSCs的寿命有限。通过进入衰老，与任何正常的体细胞40一样，MSC的增殖特征在于减少的增殖。因此，在第六次传代之前必须使用脐带血衍生的MSC，以避免细胞衰老和染色体异常41 。 为了避免这些限制，人类iPSC以高生产力为个性化，基于细胞的治疗开辟了新的可能性11 。理论上，hiPSCs的建立无限扩大。此外，与供体具有相同的免疫特性，它们可以避免在体内植入时的排斥和较低的副作用。脐带血库转为hiPSC银行同种异体药物治疗具有巨大的可能性和潜力42 。在重新编程之前筛选纯合HLA型CBMC可以广泛地利用同种异体iPSC系临床治疗。纯合子iPSC系可以避免同种异体移植后的免疫反应。该概念也可以通过产生HLA-纯合软骨11来应用于软骨再生。 通常，软骨形成分化通过两个关键步骤进行:诱导EB衍生的生长细胞和沉淀物形成。将hiPSCs初始分化为EB-衍生的生长细胞对于增加其软骨形成分化潜能至关重要。与hiPSC分化的生长细胞在功能上和分子上类似于天然BMSCs 7,43。以前的研究使用单层培养或EB培养作为诱导间充质样细胞的预分化步骤10,43。然而，与使用EBs相比，单层培养物的直接分化是耗时的<html>有代表性的颗粒倾向于分化成具有肥厚特征的纤维软骨。据报道，根据细胞单层9的细胞密度，产生的间充质样祖细胞的细胞形态和软骨形成分化潜力差异很大。 我们使用EBs来诱导生长细胞，与单层培养相比，这是一种相对快速和简单的方法。使用该协议，我们能够使用相对较少数量的hiPSC产生许多粒子（ 表1 ）。 EB的大小和数量对成功的软骨形成颗粒大量生产至关重要。因此，我们扩大了E8介质中的聚集EB，直到生成的EB的一半以上大于100μm。 EB扩大后，我们将E8培养基中的EB保持在无FGF2的状态。以前，研究人员维持生成没有FGF2的EBs用于中胚层谱系诱导外部参照"&gt; 9。 尽管我们试图将生成的生长细胞维持在高密度以获得更好的质量，但仍然存在一些限制。虽然我们已经证实产生的生长细胞具有类似的形态，但是细胞可能仍然是异源的。以前的研究尝试对特定细胞类型进行排序;然而，这个过程导致了少量细胞的收集。将需要更大规模地分离均匀的生长细胞的新尝试以产生具有增强特征的大量软骨形成颗粒。 各组通过使用单层或EB培养从hiPSC诱导祖细胞。与MSC具有高度相似性的祖细胞的诱导可能是获得更高质量的软骨形成粒细胞的关键。来自EB的这些祖细胞具有与MSC相似的特征。但是，这可能是因为成纤维细胞性因此，需要对生长细胞进行详细的验证研究。 在这项研究中，我们提出可以产生相对较大数量的软骨形成颗粒的方案。我们证实使用CBMC-hiPSCs再生的软骨表现出健康的表型，可用作组织再生的材料。然而，为了进一步应用，需要具有较短分化时间线的改进方法。用于验证具有更高水平透明质酸的软骨的质量控制标准的进一步发展也是CBMC-hiPSC作为软骨再生细胞材料的未来应用所必需的。该方案简单而有效，并且在颗粒形成之前不需要任何额外的分选过程。总之，使用该方案，可以产生高质量的软骨形成颗粒用于疾病建模，药物筛选和再生医学的研究，以进一步了解t他的软骨性质。

hiPSCs的使用代表了各种疾病的药物筛选和机械研究的新策略。从再生的角度来看，hiPSC也是替代受损组织的潜在来源，其受损愈合能力有限，如关节软骨1,2 。 天然关节软骨的再生是数十年来的挑战。关节软骨是一种柔软的白色组织，其覆盖骨骼的末端，保护其免受摩擦。然而，损坏时的再生能力有限，几乎不可能进行自我修复。因此，关注软骨再生的研究已经持续了几十年。 以前，通常使用从膝关节3分离的骨髓基质干细胞或天然软骨细胞进行体外分化成软骨形态谱系。到期o它们的软骨形成潜能，BMSCs和天然软骨细胞具有许多优点，支持它们在软骨形成中的应用。然而，由于它们的扩增和表型不稳定，这些细胞在关节软骨缺损的重建中面临着一些限制。在体外培养条件下，3-4代后，这些细胞往往失去自身特征，最终影响其分化能力。此外，在天然软骨细胞的情况下，当获得这些细胞时，对膝关节的附加损伤是不可避免的。 与BMSCs或天然软骨细胞不同，hiPSC可以在体外无限扩张。在适当的培养条件下，hiPSC作为软骨形成分化的替代来源具有巨大的潜力。然而，改变hiPSC的固有特性是很困难的。此外，它需要几个复杂的体外 steps将hiPSC的命运指向特定的单元格类型。尽管出现这些并发症，仍然推荐使用hiPSC，因为它们具有较高的自我更新能力及其分化成靶细胞的能力，包括软骨细胞6 。 软骨形成分化通常使用三维培养系统进行，例如使用MSC样祖细胞的沉淀培养或微粒培养。如果使用hiPSC，生成MSC样祖细胞的方案与现有协议不同。一些群体使用hiPSC的单层培养物将表型直接转化为MSC样细胞7 。然而，大多数研究使用EBs产生类似MSCs 8,9,10,11的生长细胞。 使用各种类型的生长因子诱导软骨NESIS。通常，单独或组合使用BMP和TGFβ家族蛋白。其他因素，如GDF5，FGF2和IGF1都有诱导分化12,13,14,15。已经显示TGFβ1在MSCs 16中以剂量依赖的方式刺激软骨形成。与其他同种型相比，TGFβ3，TGFβ1通过增加前软骨间充质细胞的冷凝来诱导软骨形成.TGFβ3通过显着增加间充质细胞增殖而诱导软骨形成。然而，TGFβ3比TGFβ17,10,18,19更频繁地被研究人员使用。 BMP2增强人类软骨形成基质成分相关基因的表达体外条件下关节软骨细胞 20 。 BMP2增加了与TGFβ蛋白21结合的MSC中软骨形成至关重要的基因表达。还已经显示BMP2通过Smad和MAPK途径协同增强TGFβ3的作用22 。 在本研究中，CBMC-hiPSC在低附着培养皿中使用EB培养基聚集成EB。通过将EB附着到明胶包被的培养皿中诱导生长细胞。使用生长细胞进行软骨形成分化。用BMP2和TGFβ3处理成功地浓缩细胞并诱导细胞外基质（ECM）蛋白质积累，用于软骨形成颗粒形成。本研究提出使用CBMC-hiPSCs的简单而有效的软骨形成分化方案。

<table><tbody><tr><td>&lt;strong&gt;Plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mm Dish</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>6-well Plate</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>50 mL Cornical Tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>15 mL Cornical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>10 mL Disposable Pipette</td><td>Falcon</td><td>7551</td><td></td></tr><tr><td>5 mL Disposable Pipette</td><td>Falcon</td><td>7543</td><td></td></tr><tr><td>12-well Plate</td><td>TPP</td><td>92012</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;E8 Medium Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12, HEPES</td><td>Life Technologies</td><td>11330-057</td><td>E8 Medium (500 mL)</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080-094</td><td>E8 Medium (Conc.: 543 &amp;mu;g/mL)</td></tr><tr><td>Sodium Selenite</td><td>Sigma Aldrich</td><td>S5261</td><td>E8 Medium&amp;nbsp; 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chondrogenic pellet formation from cord blood-derived induced pluripotent stem cells

人关节软骨缺乏修复能力。因此软骨变性不是通过治疗而是通过保守治疗来治疗。目前，正在努力用离体扩增的软骨细胞或骨髓间充质干细胞（BMSCs）再生损伤的软骨。然而，这些细胞的有限生存力和不稳定性限制了其在软骨重建中的应用。人类诱导的多能干细胞（hiPSCs）作为再生应用的新替代品已经受到科学的关注。具有无限自我更新能力和多功能性，hiPSC已被强调为软骨修复的新替代细胞来源。然而，获得高质量的软骨细胞颗粒是其临床应用的主要挑战。在本研究中，我们使用胚状体（EB）衍生的生长细胞进行软骨形成分化。通过PCR确认成功的软骨形成d染色阿片蓝，甲苯胺蓝和针对I型和II型胶原的抗体（分别为COL1A1和COL2A1）。我们提供了将脐带血单核细胞衍生的iPSC（CBMC-hiPSC）分化成软骨形成颗粒的详细方法。

在本研究中，我们通过从EBs诱导生长细胞，从CBMC-hiPSC产生软骨细胞团。使用具有确认的高多能性的CBMC-hiPSC诱导软骨形成分化11 。我们的协议的简单方案如图1A所示 。在分化前，iPSC菌落扩大（ 图1B ）。扩展的iPSC被组装为EB以启动分化（ 图1C ）。将生成的EB连接到明胶包被的培养皿以诱导生长细胞（ 图1D ）。然后，收获生长细胞并用于产生软骨形成颗粒。在分化21天后，获得小的珠状软骨形成颗粒并用于进一步表征（ 图1E ）。体外生成的质子的质子通过各种测定法证实了生殖颗粒。 我们在第21天组织学评估软骨形成颗粒的质量。使用BMSC软骨形成颗粒作为阳性对照。通过图2A中的阿尔沙蓝和甲苯胺蓝染色证实分化软骨细胞分泌的ECM蛋白的积累。随着分化过程持续21天以上，健康软骨的主要特征，如空白和蛋白多糖生产增加。由CBMC-hiPSCs产生的软骨形成颗粒表达COL2A1，其是健康软骨中的主要ECM成分（ 图2B ）。 CBMC-hiPSC衍生的颗粒的COL2A1表达高于BMSC衍生的颗粒。与COL2A1的表达相比，在CBMC-hiPSC衍生的小球中，I型胶原蛋白的表达是纤维化软骨的标志物。 </p&gt; 通过实时PCR证实21日龄软骨细胞团中软骨ECM蛋白的基因表达。来自CBMC-hiPSC的颗粒的聚集蛋白聚糖（ACAN）表达与来自BMSC的颗粒相似（ 图3A ）。 CBMC-hiPSC衍生的颗粒中COL2A1的表达显着较高（ 图3B ）。还评估了性别决定区Y盒9（Sox9）的表达，其是软骨形成的祖细胞标记物。由CBMC-hiPSCs产生的颗粒表现出高水平的Sox9（ 图3C ）。我们证实这些基因在CBMC-hiPSC软骨细胞颗粒中与第21天的BMSC对照颗粒相比显着上调。评估肥大标记COL1A1的表达（ 图3D ）。与BMSC-deri相比，CBMC-hiPSC衍生的颗粒中COL1A1的表达降低ved小丸。通过COL2A1与COL1A1的比例分析分化效率（ 图3E ）。 CBMC-hiPSC衍生的颗粒中比例的增加显示COL2A1与COL1A1相比表达量相对较高。总之，我们已经证实了CBMC-hiPSC的软骨分化能力。产生的CBMC-hiPSC衍生的软骨形成颗粒的质量与BMSCs的质量相当。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55988/55988fig1.jpg" /> 图1:hiPSC的软骨形成分化。 （ A ）hiPSCs产生软骨形成颗粒的方案。 （ B ）生成的CBMC-hiPSC的形态。 （ C ）生成的EB的形态。 （ D ）衍生自附着于明胶包被的培养皿上的EB的生长细胞。 （ E ）t的图像<html>他在21天的分化后形成软骨细胞团。间隔单位以毫米表示。刻度棒=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55988/55988fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55988/55988fig2.jpg" /> 图2:软骨形成颗粒的组织学分析。 （ A ）使用alcian蓝和甲苯胺蓝染色在第21天使用软骨细胞团的组织学评估。 （ B ）用COL1A1和COL2A1抗体染色的软骨形成细胞的免疫组织化学图像。刻度棒=100μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55988/55988fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <html> 1&quot; &gt; <img alt="图3" class="xfigimg" src="/files/ftp_upload/55988/55988fig3.jpg" /> 图3:软骨形成颗粒的基因表达。 （ A ）ACAN的相对基因表达。 （ B ）COL2A1的相对基因表达。 （ C ）SOX9的相对基因表达。 （ D ）COL1A1的相对基因表达。 （ E ）COL1A1的基因表达。 （ F ）COL10A1的基因表达。 （ G ）COL2A1:COL1A1基因表达的比例。在分化后21天收获并分析颗粒。使用实时PCR获得数据，并显示为每个样品一次性实验的平均标准误差（n = 3）。基因表达被归一化为GAPDH作为内部对照。 （* p &lt;0.05，** p &lt;0.01，*** p &lt;0.001）。k"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 手机号码 </td><td> 生长细胞产量 </td><td> 颗粒产量 </td></tr><tr><td> 人iPS细胞 </td><td> 2 x 10 6 </td><td> 2-5 x 10 7 </td><td> 70-150 </td></tr><tr><td> MSC </td><td> 2 x 10 6 </td><td> - </td><td> 6 </td></tr></tbody></table> 表1:MSC和hiPSCs的产量比较。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 目标名称 </td><td> 方向 </td><td> 引物序列 </td><td> 尺寸 </td></tr><tr><td rowspan ="2"&gt; SOX9 </td><td>前锋</td><td> TTCCGCGACGTGGACAT </td><td rowspan="2"> 77 bp </td></tr><tr><td>相反</td><td> TCAAACTCGTTGACATCGAAGGT </td></tr><tr><td rowspan="2">一个罐头</td><td>前锋</td><td> AGCCTGCGCTCCAATGACT </td><td rowspan="2"> 107 bp </td></tr><tr><td>相反</td><td> TAATGGAACACGATGCCTTTCA </td></tr><tr><td rowspan="2"> COL2A1 </td><td>前锋</td><td> GGCAATAGCAGGTTCACGTACA </td><td rowspan="2"> 79 bp </td></tr><tr><td>相反</td><td> CGATAACAGTCTTGCCCCACTTA </td></tr><tr><td rowspan="2"> COL1A1 </td><td>前锋</td><td> CCCCTGGAAAGAATGGAGATG </td><td rowspan="2"> 148 bp </td></tr><tr><td>相反</td><td> TCCAAACCACTGAAACCTCTG </td></tr></tbody></table> 表2:实时PCR中针对软骨形成标记的引物序列。

Watch this Scientific Journal Video about Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

在这里，我们提出了一种软骨细胞分化从脐带血单核细胞衍生的人诱导多能干细胞的方案。

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

chondrogenic-pellet-formation-from-cord-blood-derived-induced

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.9K 次观看.  The Catholic University of Korea. 在这里，我们提出了一种软骨细胞分化从脐带血单核细胞衍生的人诱导多能干细胞的方案。 

视频: Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

生物工程

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

人工软骨植入物通过软骨内骨化进行骨再生

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

作者聚焦：通过血管化人工软骨整合增强骨再生 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

脐带血诱导的多能干细胞的软骨形成颗粒形成

摘要

探索更多视频

此视频中的章节

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

脂肪干细胞的软骨细胞分化诱导离心重力

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

分化软骨细胞与外周血源性人诱导多能干细胞

从脐带血源性多能干细胞中提取三维皮肤有机细胞

3D磁干细胞聚集和生物反应器为成熟软骨再生

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

使用间充质干细胞通过体内软骨内骨化进行整合骨形成

使用间充质干细胞通过体内软骨内骨化进行整合骨形成

使用间充质干细胞通过体内软骨内骨化进行整合骨形成

脐带血诱导的多能干细胞的软骨形成颗粒形成

摘要

探索更多视频

此视频中的章节

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

脂肪干细胞的软骨细胞分化诱导离心重力

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

分化软骨细胞与外周血源性人诱导多能干细胞

从脐带血源性多能干细胞中提取三维皮肤有机细胞

3D磁干细胞聚集和生物反应器为成熟软骨再生

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

使用间充质干细胞通过体内软骨内骨化进行整合骨形成

使用间充质干细胞通过体内软骨内骨化进行整合骨形成

使用间充质干细胞通过体内软骨内骨化进行整合骨形成