脐带血诱导的多能干细胞的软骨形成颗粒形成
1CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Division of Rheumatology, Seoul St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, 2Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, Institute of Medical Science, College of Medicine,The Catholic University of Korea
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 CBMC-iPSC一代 使用我们以前的工作23所示的协议生成CBMC-hiPSC。 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。 准备3×10 5个细胞，并在515×g和RT下离心5分…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Plasticware | |||
| 100mm Dish | TPP | 93100 | |
| 6-well Plate | TPP | 92006 | |
| 50 mL Cornical Tube | SPL | 50050 | |
| 15 mL Cornical Tube | SPL | 50015 | |
| 10 mL Disposable Pipette | Falcon | 7551 | |
| 5 mL Disposable Pipette | Falcon | 7543 | |
| 12-well Plate | TPP | 92012 | |
| Name | Company | Catalog Number | Description |
| E8 Medium Materials | |||
| DMEM/F12, HEPES | Life Technologies | 11330-057 | E8 Medium (500 mL) |
| Sodium Bicarbonate | Life Technologies | 25080-094 | E8 Medium (Conc.: 543 μg/mL) |
| Sodium Selenite | Sigma Aldrich | S5261 | E8 Medium (Conc.: 14 ng/mL) |
| Human Transfferin | Sigma Aldrich | T3705 | E8 Medium (Conc.: 10.7 μg/mL) |
| Basic FGF2 | Peprotech | 100-18B | E8 Medium (Conc.: 100 ng/mL) |
| Human Insulin | Life Technologies | 12585-014 | E8 Medium (Conc.: 20 μg/mL) |
| Human TGFβ1 | Peprotech | 100-21 | E8 Medium (Conc.: 2 ng/mL) |
| Ascorbic Acid | Sigma Aldrich | A8960 | E8 Medium (Conc.: 64 μg/mL) |
| DPBS | Life Technologies | 14190-144 | |
| Vitronectin | Life Technologies | A14700 | |
| ROCK Inhibitor | Sigma Aldrich | Y0503 | |
| Name | Company | Catalog Number | Description |
| Quality Control Materials | |||
| 18 mm Cover Glass | Superior | HSU-0111580 | |
| 4% Paraformaldyhyde | Tech & Innovation | BPP-9004 | |
| Triton X-100 | BIOSESANG | 9002-93-1 | |
| Bovine Serum Albumin | Vector Lab | SP-5050 | |
| Anti-SSEA4 Antibody | Millipore | MAB4304 | |
| Anti-Oct4 Antibody | Santa Cruz | SC9081 | |
| Anti-TRA-1-60 Antibody | Millipore | MAB4360 | |
| Anti-Sox2 Antibody | Biolegend | 630801 | |
| Anti-TRA-1-81 Antibody | Millipore | MAB4381 | |
| Anti-Klf4 Antibody | Abcam | ab151733 | |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody | Molecular Probe | A11029 | |
| Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody | Molecular Probe | A11037 | |
| DAPI | Molecular Probe | D1306 | |
| Prolong gold antifade reagent | Invitrogen | P36934 | |
| 4% Paraformaldyhyde | Tech & Innovation | BPP-9004 | |
| Tween 20 | BIOSESANG | T1027 | |
| Bovine Serum Albumin | Vector Lab | SP-5050 | |
| Anti-Collagen II antibody | abcam | ab34712 | 1:100 |
| Alcian blue | Sigma Aldrich | A3157-10G | |
| Fast Green FCF | Sigma Aldrich | F7252-25G | |
| Safranin O | Sigma Aldrich | 090m0039v | |
| Nuclear fast red | Americanmastertech | STNFR100 | |
| xylene | Duksan | 115 | |
| Ethanol | Duksan | 64-17-5 | |
| Mayer's hematoxylin solution | wako pure chemical industries | LAK7534 | |
| DAP | VECTOR LABORATORIES | SK-4100 | |
| Slide Glass, Coated | Hyun Il Lab-Mate | HMA-S9914 | |
| Trizol | Invitrogen | 15596-018 | |
| Chloroform | Sigma Aldrich | 366919 | |
| Isoprypylalcohol | Millipore | 109634 | |
| Ethanol | Duksan | 64-17-5 | |
| RevertAid First Strand cDNA Synthesis kit | Thermo Scientfic | K1622 | |
| Name | Company | Catalog Number | Description |
| Chondrogenic Differentiation Materials | |||
| DMEM | Life Technologies | 11885 | Chondrogenic media component (500 mL) |
| Penicilin Streptomycin | Life Technologies | P4333 | Chondrogenic media component (Conc.: 1 %) |
| Ascorbic Acid | Sigma Aldrich | A8960 | Chondrogenic media component (Conc.: 64 μg/mL) |
| MEM Non-Essential Amino Acids Solution (100X) | Life Technologies | 11140-050 | Chondrogenic media component (Conc.: 100 mM) |
| rhBMP-2 | R&D | 355-BM-050 | Chondrogenic media component (Conc.:100ng/ml) |
| Recombinant Hman TGF-beta3 | R&D | 243-B3-002 | Chondrogenic media component (Conc.:10ng/ml) |
| KnockOut Serum Replacement | Life Technologies | 10828-028 | Chondrogenic media component (Conc.: 1 %) |
| ITS+ Premix | BD | 354352 | Chondrogenic media component (Conc.: 1 %) |
| Dexamethasone-Water Soluble | Sigma Aldrich | D2915-100MG | Chondrogenic media component (Conc.:10-7 M) |
| GlutaMAX Supplement | Life Technologies | 35050-061 | Chondrogenic media component (Conc.: 1 %) |
| Sodium pyruvate solution | Sigma Aldrich | S8636 | Chondrogenic media component (Conc.: 1 %) |
| L-Proline | Sigma Aldrich | P5607-25G | Chondrogenic media component (40μg/ml) |
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Cite This Article
Nam, Y., Rim, Y. A., Ju, J. H. Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells. J. Vis. Exp. (124), e55988, doi:10.3791/55988 (2017).