分化软骨细胞与外周血源性人诱导多能干细胞
1Department of Orthopedics, Beijing Chao-Yang Hospital,Capital Medical University, 2Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics,Chinese Academy of Sciences, 3Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology,Tongji University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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'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）。 制备50 mL …
Representative Results
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</…
Discussion
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。?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Knockout DMEM | Invitrogen | 10829018 | Basal medium used for hiPSC culture and EB formation medium |
| Knockout Serum Replacement (KSR) | Invitrogen | 10828028 | A more defined, FBS-free medium supplement used for hiPSC culture and EB formation medium |
| Fetal bovine serum (FBS) | Hyclone | sh30070.03 | Used for hiPSC culture and EB formation medium,offers excellent value for cell culture |
| Nonessential amino acids | Chemicon | TMS-001-C | Used as a growth supplement in all the cell culture medium, to increase cell growth and viability |
| L-glutamine | Invitrogen | 35050061 | An amino acid required for cell culture |
| Basic fibroblast growth factor (bFGF) | Peprotech | 100-18B | A cytokine used for sustaining the pluripotency and self-renewal of hiPSCs |
| Dispase | Invitrogen | 17105041 | Used for hiPSC dissociation for subculture |
| DMEM | Gibco | C11960 | Basal medium used for MSC culture medium |
| 0.1% gelatin | Millipore | ES-006-B | Used for cell attachment onto the dishes |
| 0.25% trypsin/EDTA | Gibco | 25200072 | Used for cell dissociation |
| DPBS | Gibco | 14190250 | A balanced salt solution used for cell wash or reagent preparing |
| β-mercaptoethanol | invitrogen | 21985023 | Used as a growth supplement in all the cell culture medium. |
| ITS | invitrogen | 41400045 | Insulin, Transferrin, Selenium Solution.Used for chondrogenic differentiation. |
| Ascorbic acid | Sigma | 4403 | Known as vitamin C. It helps in active growth and has antioxidant property. |
| Sodium pyruvate | Gibco | 11360070 | Added to cell culture medium as an energy source in addition to glucose. |
| Transforming growth factor-beta 1 | Peprotech | AF-100-21C | A cytokine that regulate cell proliferation, growth and chondrogenic differentiation. |
| Rabbit polyclonal antibodies against Collagen II | Abcam | ab34712 | This antibody reacts with Type II collagens,which is specific for cartilaginous tissues. |
| Mouse monoclonal antibodies to Collagen X | Abcam | ab49945 | This antibody reacts with Type X collagen,which is a product of hyperthrophic chondrotocytes. |
| Permount | Fisher Scientific | SP15-100 | For mounting and long-term storage of slides |
| Toluidine blue | Sigma | 89640 | Used for proteoglycans detection. |
| Alcian blue | Amresco | #0298 | Used for glucosaminoglycans detection. |
| Papain | Sigma | P4762-25MG | Used to digest chondrogenic pellets. |
| Dimethylmethylene blue | Sigma | 341088-1G | Used to quantitate glycosaminoglyans |
| Chondroitin sulfate sodium salt from shark cartilage | Sigma | C4384-250MG | Used to draw the standard curve for sGAG content measurement. |
| Qubit dsDNA HS assay kit | Invitrogen | Q32851 (100) | Used to determine DNA content |
| TRIzol | Invitrogen | 15596018 | Used for RNA isolation from cells |
| Reverse Transcriptase System | Promega | A3500 | Used to convert RNA into cDNA |
| SYBR FAST qPCR kit Master Mix | Kapa | KK4601 | Used for Real-time PCR |
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Citer Cet Article
Li, Y., Hai, Y., Chen, J., Liu, T. Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells. J. Vis. Exp. (125), e55722, doi:10.3791/55722 (2017).