作者希望感谢张晓斌的质粒。我们也感谢王绍荣，钱前飞在实验过程中的帮助。本研究得到了国家自然科学基金（No.81101346,81271963,81100331），北京215高级人才项目（No.2014-3-025）和北京朝阳医院基金（No CYXX-2017-01）和中国科学院青年创新促进会（YL）。

从的PBC人iPS细胞的产生，该协议可以在我们先前的研究5中找到。该研究由我们机构的机构审查委员会批准。 胚胎体（EB）形成<ol><li>制备50毫升的hiPSC培养基:补充有15％敲除血清置换（KSR），5％胎牛血清（FBS），1×非必需氨基酸，55μM2-巯基乙醇，2mM L的Knockout Dulbecco&#39;s Modified Eagle培养基谷氨酰胺和8ng / mL碱性成纤维细胞生长因子（bFGF）。 </li><li>制备50 mL EB形成培养基:补充有15％KSR，5％FBS，1X非必需氨基酸，55μM2-巯基乙醇和2mM L-谷氨酰胺的DMEM。 </li><li>制备50mL基础培养基:补充有20％FBS，1×非必需氨基酸，55μM2-巯基乙醇和2mM L-谷氨酰胺的DMEM。 </li><li>准备10 mL分泌酶溶液，1 mg / mL的敲除DMEM。 </li><li>丘尔将hiPSC与饲养细胞（ 即，照射的小鼠胚胎成纤维细胞的单层）连接到60-mm组织培养皿上。当细胞80-90％融合时，分解细胞与分泌酶，并每4-5天通过hiPSCs 1:3。将细胞置于37℃和5％CO 2培养箱中。 </li><li> 当iPSCs为80-90％汇合时，使用消火玻璃针将未分化的hiPSC菌落解剖成较小的片（直径约50-100μm）。通常，使用60mm皿中的hiPSC菌落在100mm培养皿中产生EB。 <ol><li>在100毫升含10毫升EB形成培养基的非贴壁培养皿中培养小于100个小块菌落。将菜肴放入37℃和5％CO 2培养箱中。 </li></ol></li><li>每2天用等量的基础培养基代替初始培养基的约25％。倾斜的菜让EB定居。仔细取出3 mL的上层培养基中，加入4mL新鲜的基础培养基。不要打扰EB。 注意:EBs的形态特征是这些菌落，在显微镜下呈圆形，边缘光滑。 </li><li>在非贴壁培养皿中培养10天后，在37℃下，使用4mL 0.1％明胶涂覆新的100-mm组织培养皿30分钟。 </li><li>将培养基加EB从100毫米，非粘附培养皿转移到15毫升锥形管。让EBs沉淀4-5分钟。小心吸出上清液，留下少于0.5 mL的培养基加EB </li><li>将少于100个EBs种植到具有10mL基础培养基的100mm明胶包被的组织培养皿上。将菜肴放入37℃和5％CO 2培养箱中。 </li></ol>细胞颗粒形成和软骨细胞分化<ol><li>制备10mL 0.25％胰蛋白酶/乙二胺四乙酸（EDTA）。制成80毫升的碱培养基:补充有20％FBS，1×非必需氨基酸，55μM2-巯基乙醇和2mM L-谷氨酰胺的DMEM。 </li><li>制备10 mL软骨形成分化培养基:补充有10％胰岛素转铁蛋白硒溶液（ITS），0.1μM地塞米松，1mM抗坏血酸，1％丙酮酸钠和10ng / mL转化生长因子 - β1（TGF-β1）。 </li><li> 48小时后用10 mL基础培养基刷新培养基。此后，用10mL基础培养基每三天更新培养基。 注意:培养10天后，成纤维细胞生长应从EBs扩大。 <ol><li>在使用前，在37℃下将100毫升培养皿与4毫升0.1％明胶一起包衣30分钟。弃去细胞上清液并用Dulbecco的磷酸盐缓冲盐水（DPBS）洗涤细胞一次。 </li><li>用3mL 0.25％胰蛋白酶/ EDTA在37℃下将细胞消化5分钟并用4m中和L基础培养基。 </li></ol></li><li>通过上下移动5-10次将细胞分离成单个细胞，并将其通过70μm尼龙网。以200×g离心细胞悬浮液5分钟。将细胞重新种植在一个新的100毫米明胶包被的组织培养皿上，培养基与10mL基础培养基。 </li><li> 48小时后用10 mL基础培养基刷新培养基。此后，用10mL基础培养基每三天更新培养基。 注意:细胞获得均匀的成纤维细胞样形态。 </li><li> 当达到约90-100％汇合时（ 即约5-7天），用3mL 0.25％胰蛋白酶/ EDTA在37℃收获细胞5分钟。用4 mL基础培养基中和。通过上下移动5次将细胞分离成单个细胞。使用血细胞计数器来计数细胞数。 <ol><li>将3×10 5个细胞置于15mL聚丙烯管中。离心机200 xg在室温（RT）下5分钟。将细胞重新悬浮在1mL软骨形成分化培养基中。 </li></ol></li><li>以300×g将细胞重新离心3分钟，并将细胞维持在小颗粒状态。将管放入37℃和5％CO 2培养箱中21天。不要拧紧盖子并让气体更换。 </li><li>每三天用新鲜的软骨形成分化培养基代替3/4的培养基。 注意:培养21天后，应形成hiPSC软骨细胞颗粒（hiPSC-Chon）。作为阳性对照的人间充质干细胞（MSC）也被收集并在软骨形成分化培养基中培养21天以形成软骨形成颗粒（hMSC-Chon）。 </li></ol>软骨分化分析<ol><li>准备10 mL 10％中性缓冲福尔马林。 </li><li>准备50mL的0.1％阿尔申蓝试剂和50mL的1％甲苯胺蓝试剂。 </li><li>镨表达1毫升的一级抗体:针对胶原II（1:50）的兔多克隆抗体或针对胶原X的小鼠单克隆抗体（1:50）。另外，制备抗兔或小鼠二次抗体。 </li><li>在含有0.1M乙酸钠，2.4mM EDTA和5mM L-半胱氨酸的PBS中制备1mL木瓜蛋白酶溶液:10U / mL。 </li><li>制备100mL二甲基亚甲基蓝（DMMB）染料溶液:100μL的16mg / L的1,9-二甲基亚甲蓝，40mM的甘氨酸，40mM的NaCl和9.5mM的HCl; pH 3.0。 </li><li> 评估粒细胞部分的阿隆蓝和甲苯胺蓝染色体的软骨形成分化。 <ol><li>将一个hiPSC-Chon颗粒或hMSC-Chon颗粒固定在1 mL 10％中性缓冲福尔马林中24 h。 </li><li>将沉淀物转移到1mL 70％乙醇的H 2 O中。用1mL分级乙醇系列（ 即 25,50,75,90,95,100和100％，每次3分钟）脱水沉淀。 </li><li>将沉淀物在1mL 100％二甲苯中澄清三次。 Infiltrat在65℃烘箱中用石蜡包埋1小时。按照常规组织学步骤6将颗粒嵌入具有7×7×5mm 3基底模具的石蜡块中。 </li><li>使切片机由相邻的部分的厚度为大约4微米7。将部分粘贴在玻璃片上。 </li><li>在60℃下在烤箱中干燥载玻片2小时。使用100％二甲苯将三个循环（每次3分钟）进行脱蜡。 </li><li>使用减少的酒精系列进行再水化（ 即， 100，100，95，95，70，50和25％，在H 2 O中，每次3分钟），然后用去离子水进行最终冲洗5分钟。 </li><li>用0.1％阿隆蓝色试剂或1％甲苯胺蓝染色染色4-5小时，然后用蒸馏水冲洗。 </li><li>用分级乙醇系列（ 即 25,50,75,90,95,100和100％，每个3分钟）脱水，然后连续三次在100％二甲苯中澄清的eps。安装幻灯片并在显微镜下可视化。 </li></ol></li><li> 进行免疫组化。 注意:通过免疫组织化学进一步评估额外的颗粒切片。 <ol><li>脱蜡和再水合后，将载玻片在1mM EDTA，pH8.0（沸水煮沸的水中防水）中沸腾。让它们在亚沸点温度下静置8分钟，然后让载玻片在室温下冷却。 </li><li>用去离子水冲洗载玻片3次。在室温下将3％H 2 O 2溶液在甲醇中孵育15分钟以阻断内源性过氧化物酶活性。 </li><li>用去离子水冲洗载玻片并将其浸入DPBS中5分钟。应用50-100μL适当稀释（1:50）的一抗，在载玻片上切片，然后在室温下将其孵育1 h。 </li><li>用DPBS洗涤载玻片3次（每次5分钟）。孵化sa与相应的二抗（ 即抗兔或小鼠）在室温下混合15分钟。 </li><li>用DPBS洗涤载玻片3次（每次5分钟）。在显微镜下进行DAB检测。 </li><li>用DPBS洗涤载玻片3次（每次2分钟）。通过将载玻片浸入苏木精中1-2分钟来复制细胞核。用分级乙醇系列（25,50,75,90,95,100和100％;每次3分钟）脱水，然后用二甲苯澄清三个连续步骤。最后，安装幻灯片并在显微镜下可视化。 </li></ol></li><li> 检测sGAG内容。 <ol><li>在木瓜蛋白酶溶液中在60℃下将消化软骨细胞沉淀2小时。 </li><li>使用dsDNA测定试剂盒和荧光计系统确定DNA含量。通过在525nm处测量吸光度下与DMMB染料溶液混合来测量sGAG含量8 。 </li><li>根据标准曲线计算sGAG的浓度鲨鱼硫酸软骨素</li></ol></li><li> 对hiPSC-Chon颗粒中的软骨形成分化标记进行实时PCR分析。 <ol><li>通过向一个管中加入500μL冰冷的提取试剂，收获3-4个相同的软骨形成颗粒。彻底旋涡在室温下孵育样品5分钟。 </li><li>加入0.1mL氯仿。将样品涡旋15秒，并在室温下孵育3分钟。在12,000 xg和4℃下将样品离心15分钟。将上层水相（约250μL）转移到新的1.5 mL微量离心管中。 </li><li>向样品中加入25μL乙酸钠和1μL糖原。将RNA与250μL异丙醇混合，沉淀RNA。彻底混合在室温下孵育样品10分钟。 </li><li>在12,000 xg和4℃下离心10分钟。彻底清除上清液。用500μL的75％乙醇洗涤RNA沉淀两次。 </li><li>离心5分钟在7,500 xg和4°C。取出所有剩余的乙醇，并将RNA颗粒空气干燥5-10分钟。将RNA溶解在10μL无核酸酶的水中。 </li><li>使用逆转录酶系统将RNA转化为cDNA。使用qPCR试剂盒主混合物（2x）和实时PCR系统对cDNA样品进行实时PCR 9 。 注:引物序列为: h AGGRECAN -F:TCGAGGACAGCGAGGCC; h AGGRECAN -R:TCGAGGGTGTAGCGTGTAGAGA; hβ -ACTIN- F:TTTGAATGATGAGCCTTCGTCCCC; hβ -ACTIN -R:GGTCTCAAGTCAGTGTACAGGTAAGC; h COL2- F:TGGACGATCAGGCGAAACC; h COL2 -R:GCTGCGGATGCTCTCAATCT; h SOX9- F:AGCGAACGCACATCAAGAC; h SOX9 -R:CTGTAGGCGATCTGTTGGGG; h COL10 -F:ATGCTGCCACAAATACCCTTT; h COL10 -R:GGTAGTGGGCCTTTTATGCCT。 </li></ol></li></ol>

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作者没有什么可以披露的。

在这里，我们提供了通过iPSC从PBCs产生软骨细胞的方案。因为PBC在临床领域更常见并被广泛使用，所以它们被认为是重新编程的潜在替代方案。在本研究中，利用附加型载体（EV）将PBC重编程为iPSC，按照Zhang 等人建立的方法11 。这个免费的集成的方法不涉及整合型病毒相关的遗传毒性，这被认为是在临床领域12，13广阔的效果。本研究满足了从血细胞中产生无集成iPSCs的重新编程效率。超过30个iPSC可以从2毫升的外周血中产生。因此，PBC有可能成为用于产生软骨工程和其他临床应用的iPSC的种子细胞。 软骨形成的主要步骤不同来自hiPSCs的结合包括:EB形成，EBs细胞增殖，单层培养和3D沉淀培养。使用消防玻璃针将未分化的hiPSC菌落解剖成较小的片段。机械方法虽然比技术更好，但由于损伤和特定尺寸（直径为50-100μm）在采集时比酶消化（如分解酶或胶原酶）更好。此外，机械消化可以手动处理饲养细胞，这将抑制hiPSC分化。 hiPSCs自发分化形成EB，其特征在于具有光滑边界的三维多细胞聚集体。几个EB可以聚集在一起形成不规则形状。为了保持EB在良好的条件下，在100毫米非粘附培养皿中培养少于100个EB，并加入10毫升EB形成培养基。一个100毫米盘中的约50只EB被认为是最好的集中。然后将EB接种<html>至10厘米，具有基础培养基的明胶包被的培养皿。 EB的分布密度对EB的足够长的生长是重要的。少于100个EB被培养在100毫米的盘子上。培养10天后，成纤维细胞逐渐长出并从EB扩增出来。执行单层步骤以排除存在于EB中的残留未分化细胞，以及扩展致力于间充质谱系的细胞。将0.5-1×10 6个细胞接种在用于单层细胞培养的100-mm培养皿中。对的hiPSC-F细胞表面标志物的表达，通过流式细胞分析，我们在以前的研究中分析了5。结果表明，大多数hiPSC-F表达CD73（81.81±2.05％）和CD105（内皮糖蛋白; 81.90±1.61％），已知它们是阳性人间质标志物。此外，已经使用六种不同的iPSC和一种人胚胎干细胞（ESC）来再现这些方法。 多能细胞的诱导软骨形成分化也是一个复杂的关键过程，鉴于此，经典软骨形成培养基用于诱导hiPSCs的软骨形成，TGF-β1和地塞米松在沉淀培养基中补充，这些因子已被证明对软骨形成潜能14显著的影响。从其它的协议的另一种差异是10％ITS的浓度，其比典型地报告了1％ITS 15,16。1％ITS加10％FBS增强高得多软骨形成的其它方法17,18。ITS作为血清替代品可促进软骨细胞的增殖和形成和保持软骨的表型。为了更换FBS的动物成分，我们升级的ITS至10％的浓度，这已被证明有效地推广软骨细胞分化7 。 高密度细胞培养是成软骨分化的另一个重要因素。存在可用于诱导软骨形成分化许多其它细胞培养方法，如微团培养，共培养与其它细胞，基于生物材料培养，和遗传操作1，19，15。我们研究中导致高细胞密度和高细胞 - 细胞相互作用的3D沉淀培养在没有其他细胞或材料的情况下更容易进行。由于它是在15 mL离心管中进行的，一个限制是它只能用于小规模成软骨分化测定。然而，具有圆底的96孔板可以用作有希望的替代品7 。因此，培养方法的其他改善可以促进软骨形成的效率体外分化 。在我们的研究中，长达21天的软骨形成诱导在无血清和不含异种病毒条件下进行，在此期间除去所有与动物相关的成分。因此，本研究中的手术适用于未来的临床应用。 据认为，自体干细胞将是软骨修复的理想选择，因为它们不仅可以减少排斥反应，而且还通过采取胚胎发育20,21的自然病程的优势实现组织再生。然而，他们发现在体外具有有限的增殖潜力22 。因此，通过iPSCs从PBCs产生软骨细胞的无整合方法可能是软骨组织工程更有前途的方法。用我们的方法，2毫升血液可以足以诱导软骨脱屑所需的患者特异性软骨细胞TS。此外，我们还使用hMSCs作为阳性对照，与从iPSC分化的细胞进行比较，这表明iPSC具有良好的软骨形成分化潜能。 总之，这项研究证明PBCs可以作为软骨细胞再生的候选者。这可以进一步反映出用于以再生医学的患者特异性和成本有效的方法产生用于软骨修复的种子细胞的未来方向。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55722/differentiating-chondrocytes-from-peripheral-blood-derived-human">Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells</a>.
The corresponding author was updated from:
Yueying Li 
yueyinglee2010@hotmail.com 
Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences
The corresponding author is now listed as:
Tie Liu 
waterkins@163.com 
Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55722/differentiating-chondrocytes-from-peripheral-blood-derived-human">Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells</a>.

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

软骨组织的自我修复和再生能力非常差。使用各种手术干预和生物处理来恢复软骨和关节功能，结果令人不满。干细胞技术的最新发展可能会改变整个软骨修复领域1 。已经研究了各种干细胞作为种子细胞，但人诱导的多能干细胞（hiPSC）似乎是最有希望的选择，因为它们可以提供许多类型的患者特异性细胞而不引起排斥反应2 。此外，他们可以克服成人细胞的增殖性有限，维持自我更新和多能力。此外，基因靶向可用于改变基因型以获得特定类型的软骨细胞。 成纤维细胞已被广泛用于产生iPSC，因为它们的重编程潜力也得到了很好的研究。然而，还有一些必须克服的局限性，例如患者的疼痛活检和成纤维细胞的体外扩增的需要，这可能导致基因突变3 。最近，PBC被发现有利于重编程4 ;此外，它们被普遍利用并且被大量储存。它们可能会将研究重点从皮肤重定向。然而，据我们所知，关于PBC重新编程，随后分化成软骨细胞的报道很少。 在本研究中，我们将PBCs作为替代来源，通过将其重新编程到iPSC中，然后通过沉淀培养系统将iPSC分化成软骨细胞系，以模拟软骨细胞形成。

<table><tbody><tr><td>Knockout DMEM</td><td>Invitrogen</td><td>10829018</td><td>Basal medium used for hiPSC culture and EB formation medium</td></tr><tr><td>Knockout Serum Replacement (KSR)</td><td>Invitrogen</td><td>10828028</td><td>A more defined, FBS-free medium supplement used for hiPSC culture and EB formation medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>sh30070.03</td><td>Used for hiPSC culture and EB formation medium,offers excellent value for cell culture</td></tr><tr><td>Nonessential amino acids</td><td>Chemicon</td><td>TMS-001-C</td><td>Used as a growth supplement in all the cell culture medium, to increase cell growth and viability</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>TMS-002-C</td><td>An amino acid required for cell culture</td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Peprotech</td><td>100-18B</td><td>A cytokine used for sustaining the pluripotency and self-renewal of hiPSCs</td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105041</td><td>Used for hiPSC dissociation for subculture</td></tr><tr><td>DMEM</td><td>Gibco</td><td>C11960</td><td>Basal medium used for MSC culture medium</td></tr><tr><td>0.1% gelatin</td><td>Millipore</td><td>ES-006-B</td><td>Used for cell attachment onto the dishes</td></tr><tr><td>0.25% trypsin/EDTA</td><td>Gibco</td><td>25200072</td><td>Used for cell dissociation</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190250</td><td>A balanced salt solution used for cell wash or reagent preparing</td></tr><tr><td>2-mercaptoethanol</td><td>invitrogen</td><td>21985023</td><td>Used as a growth supplement in all the cell culture medium.</td></tr><tr><td>ITS</td><td>invitrogen</td><td>41400045</td><td>Insulin, Transferrin, Selenium Solution.Used for chondrogenic differentiation.</td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>4403</td><td>Known as vitamin C. It helps in active growth and has antioxidant property.</td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360070</td><td>Added to cell culture medium as an energy source in addition to glucose.</td></tr><tr><td>Transforming growth factor-beta 1</td><td>Peprotech</td><td>AF-100-21C</td><td>A cytokine that regulate cell proliferation, growth and chondrogenic differentiation.</td></tr><tr><td>Rabbit polyclonal antibodies against Collagen II</td><td>Abcam</td><td>ab34712</td><td>This antibody reacts with Type II collagens,which is specific for cartilaginous tissues.</td></tr><tr><td>Mouse monoclonal antibodies to Collagen X</td><td>Abcam</td><td>ab49945</td><td>This antibody reacts with Type X collagen,which is a product of hyperthrophic chondrotocytes.</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-100</td><td>For mounting and long-term storage of slides</td></tr><tr><td>Toluidine blue</td><td>Sigma</td><td>89640</td><td>Used for proteoglycans detection.</td></tr><tr><td>Alcian blue</td><td>Amresco</td><td>#0298</td><td>Used for glucosaminoglycans detection.</td></tr><tr><td>Papain</td><td>Sigma</td><td>P4762-25MG</td><td>Used to digest chondrogenic pellets.</td></tr><tr><td>Dimethylmethylene blue</td><td>Sigma</td><td>341088-1G</td><td>Used to quantitate glycosaminoglyans</td></tr><tr><td>Chondroitin sulfate sodium salt from shark cartilage</td><td>Sigma</td><td>C4384-250MG</td><td>Used to draw the standard curve for sGAG content measurement.</td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Invitrogen</td><td>Q32851 (100)</td><td>Used to determine DNA content</td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596018</td><td>Used for RNA isolation from cells</td></tr><tr><td>Reverse Transcriptase System</td><td>Promega</td><td>A3500</td><td>Used to convert RNA into cDNA</td></tr><tr><td>SYBR FAST qPCR kit Master Mix</td><td>Kapa</td><td>KK4601</td><td>Used for Real-time PCR</td></tr></tbody></table>

differentiating chondrocytes from peripheral blood-derived human induced pluripotent stem cells

在本研究中，我们使用外周血细胞（PBC）作为种子细胞，通过诱导多能干细胞（iPSCs）以无积分方法产生软骨细胞。在胚状体（EB）形成和成纤维细胞扩增后，在无血清和无异种病毒条件下诱导iPSC进行软骨形成分化21天。在软骨细胞诱导后，通过形态学，免疫组织化学和生物化学分析以及软骨形成分化标志物的定量实时PCR检测来评价细胞的表型。软骨细胞团块显示阳性阿an蓝和甲苯胺蓝染色。胶原蛋白II和X染色的免疫组化也是阳性的。硫酸化糖胺聚糖（sGAG）含量和软骨形成分化标记COLLAGEN 2 （ COL2 ）， COLLAGEN 10 （ COL10 ）， SOX9和AGGRECAN在chond中显着上调与hiPSCs和成纤维细胞相比，生殖球粒。这些结果表明，PBCs可用作种子细胞以产生用于软骨修复的iPSC，其是患者特异性且具有成本效益的。

hiPSCs的软骨形成分化: 使用EB形成培养基和基础培养基将hiPSC分化为间充质谱系。使用多步培养方法（ 图1 ）。首先，通过EB形成自发分化hiPSCs 10天（D10; 图2A ）。其次，细胞从EBs中延续10天（D10 + 10）。在这两个步骤中，iPSCs逐渐失去原始形态并获得纺锤形形态（ 图2B ），然后在通过后变为成纤维细胞形状。第三，细胞在传代培养后以单层扩张（ 图2C ）。在此步骤中排除残留的未分化细胞。然后，细胞扩大并致力于成纤维细胞样细胞单层培养5-10天（D10 + 10 + 7）。第四，当hiPSC-成纤维细胞样细胞（hiPSC-F）达到约90％汇合时，通过3D沉淀培养（ 图2D ） 10诱导它们分化成软骨细胞。 hiPSC-Chon颗粒的表征: 将hiPSC-F在沉淀的15mL聚丙烯管中培养21天。软骨形成细胞可以在体外组装，并在高密度培养时产生特征性细胞外基质。在培养结束时，我们可以看到密实的软骨样聚集体，即高达2-3毫米长和3毫米厚的hiPSC-Chon颗粒（ 图2D ）。细胞对alcian蓝色（ 图3A ）和甲苯胺蓝（ 图3B ）染色呈阳性，表明成功ul软骨细胞分化为hiPSC颗粒。胶原II（ 图3C ）和胶原X（ 图3D ）的免疫组织化学分析进一步证实了hiPSC-Chon颗粒已经形成软骨细胞样表型。进行胶原II和胶原X的免疫组织化学的阴性对照以更好地证实阳性染色（数据未显示） 5 。 软骨分化后也进行sGAG分析（ 图3E ）。在hiPSC-Chon颗粒，hiPSC-F，EBs和未分化的hiPSC中检测到sGAG含量。 sGAG含量在hiPSC-Chon颗粒中显着高于其他组（ P &lt;0.05）。在hMSC-Chon的阳性对照中，与hMSC相比，sGAG含量也显着上调（ P &lt;0.05）。但是，sGAG的内容hMSC颗粒和hiPSC-Chon颗粒之间没有差异（ P &gt; 0.05）。 软骨分化标记的基因表达: 使用软骨祖细胞谱系（ SOX9和COL2 ）和完全分化软骨细胞（ AGGRECAN和COL10 ）的分化标志物的基因表达来表征软骨形成颗粒的表型（ 图4 ）。在hiPSC，hiPSC-F和hiPSC-Chon颗粒之间的比较中，hiPSC-Chon中COL2 ， COL10 ， SOX9和AGGRECAN的表达显着上调，而其他组（ P &lt;0.05）。在hMSC-Chon的阳性对照中，这些标记物的表达也显着高于hMSC（ P &lt;0.05）。然而，hMS之间的基因表达C颗粒和hiPSC-Chon颗粒没有差异（ P &gt; 0.05）。总之，这些结果表明人类iPSCs成功的成软骨分化过程。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55722/55722fig1.jpg" /> 图1:协议示意图。一种用于将人iPSC分化为软骨细胞的多步培养方法，包括: 1） 通过 EB形成自发分化，2）EBs的细胞生长，3）传代培养后的单层细胞培养，以及4）3D沉淀培养。通过组织学分析，生物化学分析和软骨形成基因表达来评估软骨细胞表型。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55722/55722fig1large.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 / 55722 / 55722fig2.jpg"/&gt; 图2:来自hiPSC的软骨细胞的产生。 （ A ）D10上的EB形成。刻度棒=100μm。 （ B ）D10 + 10上EBs细胞生长。刻度棒=100μm。 （ C ）D10 + 10 + 7上的单层细胞培养。刻度棒=100μm。 （ D ）3D颗粒培养。这个数字已经从我们以前的研究5修改了。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55722/55722fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55722/55722fig3.jpg" /> 图3:hiPSC-Chon颗粒的表征。 （ A ） 阿蓝染色和（ B ）甲胺蓝染色的糖胺聚糖和蛋白酶聚糖。刻度棒=100μm。 （ C和D ）胶原II和胶原X的免疫组织化学。比例尺=100μm。 （ E ）与hMSC-Chon与hMSC相比，hiPSC-Chon颗粒与hiPSC，EB和hiPSC-F的生物化学表征。 sGAG每个DNA。棒表示平均值±SEM。 N = 3，* P &lt;0.05。这个数字已经从我们以前的研究5修改了。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55722/55722fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55722/55722fig4.jpg" /> 图4:基因表达分析。 hiPSC-Chon对hiPSC和hiPS中软骨形成分化标志物（ COL2 ， COL10 ， SOX9 和AGGRECAN ）的RT-qPCR基因表达分析CF与hMSC-Chon与hMSC相比。棒表示平均值±SEM。 N = 3，* P &lt;0.05。这个数字已经从我们以前的研究5修改了。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55722/55722fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells at JoVE.com

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

我们提出了一种通过诱导多能干细胞（iPSCs）从人外周血（PB）产生软骨形态谱的方案，使用无积分法，其包括胚状体（EB）形成，成纤维细胞扩增和软骨形成诱导。

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 ...

differentiating-chondrocytes-from-peripheral-blood-derived-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  Capital Medical University. 我们提出了一种通过诱导多能干细胞（iPSCs）从人外周血（PB）产生软骨形态谱的方案，使用无积分法，其包括胚状体（EB）形成，成纤维细胞扩增和软骨形成诱导。 

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

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 ...

科研

发育生物学

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

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

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 ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

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 ...

生物工程

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

生物学

表征 hiPSC 驱动 MSC 中的表面抗原和增殖能力

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

作者聚焦:iPSC 和遗传病研究的进展 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

分化软骨细胞与外周血源性人诱导多能干细胞

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此视频中的章节

脂肪干细胞的软骨细胞分化诱导离心重力

人多能干细胞对原始和最终造血祖细胞定向分化的研究

脐带血诱导的多能干细胞的软骨形成颗粒形成

从脐带血源性多能干细胞中提取三维皮肤有机细胞

血源性内皮细胞与人多能干细胞的定向分化

破骨细胞与人诱导多能干细胞的分化和表征

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

人主动脉周脂肪祖细胞的分化能力

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较