高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化
Summary
本协议介绍了一种简单的两步法在异种和无细胞培养条件下, 从人多潜能干细胞中分化角膜缘上皮干细胞的方法。这里介绍的细胞培养方法可实现经济高效的大规模生产, 适用于角膜细胞治疗使用的临床质量细胞。
Abstract
角膜缘上皮干细胞 (LESCs) 负责持续更新角膜上皮细胞, 从而保持角膜的稳态和视觉清晰度。人类多能干细胞 (hPSC) 衍生 LESCs 为角膜细胞置换治疗提供了有希望的细胞来源。未定义、异种的培养和分化条件导致了研究结果的变化, 阻碍了 hPSC 治疗的临床翻译。该协议为异种和馈线无细胞条件下的 hPSC LESC 分化提供了一种可重现和有效的方法。首先, 对重组 laminin-521 (LN-521) 和定义的 hPSC 培养基中未分化 hPSC 的单层培养是稳定生产高质量起始材料的基础。其次, 快速简单的 hPSC LESC 分化方法仅在24天内就会产生 LESC 种群。该方法包括四天表面外胚层诱导的小分子悬浮液, 其次是在 LN-521/collagen IV 组合基质上贴壁培养相, 确定角膜上皮分化培养基。Cryostoring 和扩展分化进一步净化细胞的种群, 并使细胞的银行在大量的细胞治疗产品。由此产生的高质量 hPSC LESCs 为角膜表面重建治疗角膜缘干细胞缺乏 (康乐文化署) 提供了一种潜在的新的处理策略。
Introduction
眼睛表面的透明角膜允许光线进入视网膜并提供大部分眼球的折射力。角膜缘上皮干细胞 (LESCs) 持续再生, 是最外层的层状角膜上皮细胞。LESCs 位于巩膜交界处1,2的角膜缘壁龛的基底层。LESCs 缺乏特定的和独特的标记, 因此它们的识别需要对一套假定标记进行更广泛的分析。上皮转录因子 p63, 特别是 p63 (ΔNp63α) 阿尔法异构体的 n-末期截断转录, 已被提出作为一个相关的阳性 LESC 标记3,4。LESCs 的不对称分裂允许他们自我更新, 但也产生迁移向心地和前方的后代。随着细胞向角膜表面的进展, 他们逐渐失去了干性, 最后最终分化为从角膜表面不断丢失的表面鳞状细胞。
对任何角膜层的损伤都会导致严重的视力损害, 因此角膜缺陷是全球视力丧失的主要原因之一。在角膜缘干细胞缺乏症 (康乐文化署), 角膜缘被破坏的疾病或创伤导致 conjunctivalization 和白内障的表面和随后的视力损失5,6。使用自体或异基因角膜缘移植的细胞置换疗法为康乐文化署4、7、8、9的患者提供治疗策略。然而, 收获自体移植有并发症的风险, 健康的眼睛, 捐助组织是供不应求。人类多能干细胞 (hPSCs), 特别是人类胚胎干细胞 (hESCs) 和人类诱导的多能干细胞 (hiPSCs), 可以作为一种无限的临床相关细胞类型, 包括角膜上皮细胞的来源。因此, hPSC 衍生的 LESCs (hPSC LESCs) 代表了一种吸引眼球细胞替代疗法的新细胞源。
传统上, 未分化的 hPSC 培养方法和 LESCs 的分化协议依赖于未定义的馈线细胞、动物血清、条件培养基或羊膜10、11、12,13,14,15. 最近, 对更安全的细胞治疗产品的努力促使人们寻求更标准化和异种的文化和差异化协议。因此, 对未分化 hPSCs 的长期培养的几种定义和异种的方法现在在商业上可用16,17,18。作为一个连续的, 定向分化协议依赖分子线索引导 hPSCs 角膜上皮的命运最近介绍了19,20,21,22, 23。然而, 许多这些协议使用的是未定义的, 以馈线为基础的 hPSCs 作为起始材料, 或复杂, 异种生长因子鸡尾酒的分化。
本协议的目的是提供一个强大的, 优化的, 异种和无饲养的 hPSC 培养方法和随后分化的角膜 LESCs。多能 hPSCs 在 laminin-521 (LN-521) 基质中的单层培养在定义的无白蛋白 hPSC 培养基 (特别重要的 8 Flex) 允许快速生产均匀起始材料的分化。此后, 一个简单的, 两步分化策略引导 hPSCs 朝向表面外胚层的命运在悬浮, 其次是贴壁分化到 LESCs。在24天内获得 > 65% express ΔNp63α的细胞种群。异种和无馈线协议已通过几个 hPSC 线 (hESCs 和 hiPSCs) 进行了测试, 没有任何对细胞系特定优化的要求。此处介绍的无周末维护、传代、cryostoring 和 hPSC LESC 分型协议可为临床或研究目的生产大量高质量 LESCs。
Protocol
坦佩雷大学经芬兰国家法医学鉴定事务局 (Dnro 1426/32/300/05) 批准, 对人类胚胎进行研究。该研究所还有皮尔卡曼医院区道德委员会的支持性声明, 以推导、培养和区分人类胚胎干细胞线 (Skottman/R05116), 并在眼科研究中使用 hiPSC 线 (Skottman/R14023)。这项研究没有新的细胞系。 注意: 所述的协议是基于特定的, 商业上可用的 hPSC 和角膜上皮分化培养基。有关制造商/供应商信息和目录编…
Representative Results
从 hPSCs 到 hPSC-LESCs 从诱发 FF hPSCs 到 cryostoring hPSC LESCs 的整个过程大约需要3.5 周。图 1A给出了突出其关键步骤的微分方法的原理图概述。图 1B显示了协议不同阶段细胞种群的典型形态。所提供的数据采用 Regea08/017 人类胚胎干细胞线和 04607. WT hiPSC 线, 在芬兰坦佩雷大学获得和表征, 如?…
Discussion
该协议的预期结果是在大约3.5 周内从 FF hPSC 的单个电池悬浮液中成功和稳健地生成 LESCs。由于角膜上皮发育从表面外胚层29, 该议定书的第一步旨在指导 hPSCs 走向这一血统。24 h 诱导与转化生长因子β (SB-505124) 拮抗剂, 和 bFGF 用于诱导外胚层分化, 其次 48 h 中胚层 BMP-4 提示推动细胞朝向表面外胚层。将 IV/LN-521 组合矩阵的以下贴壁分化步骤与化学定义的分化培养基结合在一起, 进?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该研究得到了芬兰科学院 (赠款297886号)、特克斯的人力备件计划、芬兰技术和创新资助机构、芬兰眼和组织银行基金会以及芬兰文化基金会的支持。作者感谢生物医学实验室技术人员我 Melin、汉娜 Pekkanen、艾玛维克斯泰特和我 Heikkilä为卓越的技术援助和对细胞培养的贡献。Katriina 教授 Setälä被公认为提供用于荧光成像设备的 hiPSC 线和普司通成像核心设施。
Materials
| Material/Reagent | |||
| 1x DPBS containing Ca2+ and Mg2+ | Gibco | #14040-091 | |
| 1x DPBS without Ca2+ and Mg2+ | Lonza | #17-512F/12 | |
| 100 mm cell culture dish | Corning CellBIND | #3296 | Culture vessel format for adherent hPSC-LESC differentiation |
| 12-well plate | Corning CellBIND | #3336 | Culture vessel format for IF samples |
| 24-well plate | Corning CellBIND | #3337 | Culture vessel format for IF samples |
| 2-mercaptoethanol | Gibco | #31350-010 | |
| 6-well plate, Ultra-Low attachment | Corning Costar | #3471 | Culture vessel format for induction in suspension culture |
| Alexa Fluor 488 anti-mouse Ig | ThermoFisher Scientific | #A-21202 | Secondary antibody for IF |
| Alexa Fluor 488 anti-rabbit Ig | ThermoFisher Scientific | #A-21206 | Secondary antibody for IF |
| Alexa Fluor 568 anti-goat Ig | ThermoFisher Scientific | #A-11057 | Secondary antibody for IF |
| Alexa Fluor 568 anti-mouse Ig | ThermoFisher Scientific | #A-10037 | Secondary antibody for IF |
| Basic fibroblast growth factor (bFGF, human) | PeproTech Inc. | #AF-100-18B | Animal-Free Recombinant Human FGF-basic (154 a.a.) |
| BD Cytofix/Cytoperm Fixation/Permeabilization Solution | BD Biosciences | #554722 | Fixation and permeabilization solution for flow cytometry |
| BD Perm/Wash Buffer | BD Biosciences | #554723 | Washing buffer for flow cytometry |
| Blebbistatin | Sigma-Aldrich | #B0560 | |
| Bone morphogenetic protein 4 (BMP-4) | PeproTech Inc. | #120-05A | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | #A8022-100G | |
| Cytokeratin 12 antibody | Santa Cruz Biotechnology | #SC-17099 | Primary antibody for IF |
| Cytokeratin 14 antibody | R&D Systems | #MAB3164 | Primary antibody for IF |
| Cytokeratin 15 antibody | ThermoFisher Scientific | #MS-1068-P | Primary antibody for IF |
| CnT-30 | CELLnTEC Advanced Cell Systems AG | #Cnt-30 | Culture medium for adherent hPSC-LESC differentiation |
| Collagen type IV (human) | Sigma-Aldrich | #C5533 | Human placental collagen type IV |
| CoolCell LX Freezing Container | Sigma-Aldrich | #BCS-405 | |
| CryoPure tubes | Sarsted | #72.380 | 1.6 ml cryotube for hPSC-LESC cryopreservation |
| Defined Trypsin Inhibitor | Gibco | #R-007-100 | |
| Essential 8 Flex Medium Kit | Thermo Fisher Scientific | #A2858501 | |
| GlutaMAX | Gibco | #35050061 | |
| Laminin 521 | Biolamina | #Ln521 | Human recombinant laminin 521 |
| ΔNp63α antibody | BioCare Medical | #4892 | Primary antibody for IF |
| OCT3/4 antibody | R&D Systems | #AF1759 | Primary antibody for IF |
| p63α antibody | Cell Signaling Technology | #ACI3066A | Primary antibody for IF |
| p63-α (D2K8X) XP Rabbit mAb (PE Conjugate) | Cell Signaling Technology | #56687 | p63-α PE-conjugated antibody for flow cytometry |
| PAX6 antibody | Sigma-Aldrich | #HPA030775 | Primary antibody for IF |
| Penicillin/Streptomycin | Lonza | #17-602E | |
| Paraformaldehyde (PFA) | Sigma-Aldrich | #158127 | Cell fixative for IF |
| ProLong Gold Antifade Mountant with DAPI | Thermo Fisher Scientific | #P36931 | DAPI mountant for hard mounting for IF |
| PSC Cryopreservation Kit | Thermo Fisher Scientific | #A2644601 | |
| TrypLE Select Enzyme | Gibco | #12563-011 | |
| KnockOut Dulbecco’s modified Eagle’s medium | Gibco | #10829018 | |
| KnockOut SR XenoFree CTS | Gibco | #10828028 | |
| MEM non-essential amino acids | Gibco | #11140050 | |
| SB-505124 | Sigma-Aldrich | #S4696 | |
| Triton X-100 | Sigma-Aldrich | #T8787 | Permeabilization agent for IF |
| VectaShield | Vector Laboratories | #H-1200 | DAPI mountant for liquid mounting for IF |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Cytocentrifuge, e.g. CellSpin II | Tharmac | ||
| Flow cytometer, e.g. BD Accuri C6 | BD Biosciences | ||
| Fluorescence microscope, e.g.Olympus IX 51 | Olympus |
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Cite This Article
Hongisto, H., Vattulainen, M., Ilmarinen, T., Mikhailova, A., Skottman, H. Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells. J. Vis. Exp. (140), e58279, doi:10.3791/58279 (2018).