人多能干细胞对原始和最终造血祖细胞定向分化的研究
Summary
在这里, 我们提出了人类多能干细胞 (hPSC) 的文化协议, 用于区分 hPSCs 成 CD34 的+造血祖细胞。此方法使用特定于阶段的规范 WNT 信号的操作来指定单元格完全是最终的或原始的造血程序。
Abstract
再生医学的主要目标之一是从人类多能干细胞 (hPSCs) 中产生和维持造血干细胞 (干细胞)。直到最近, 将 hPSCs 分化为干细胞的努力主要产生了缺乏 HSC 潜能的造血祖细胞, 而不是像卵黄囊造血。这些产生的造血祖细胞可能有有限的效用, 为体外疾病模型的各种成人造血疾病, 特别是那些淋巴谱系。然而, 我们最近描述的方法产生赤-骨髓淋巴多向确定造血祖细胞的 hPSCs 使用一个阶段特定的定向分化协议, 我们在这里概述。通过在基底膜 plasticware 上 hPSCs 酶解离, 形成了胚体 (EBs)。EBs 被区分对胚层由重组 BMP4, 随后被指定到明确的造血程序由 GSK3β抑制剂, CHIR99021。另外, 原始的造血是由 PORCN 抑制剂, IWP2 指定的。造血是进一步推动通过增加重组 VEGF 和支持造血细胞因子。使用这种方法产生的造血祖细胞有可能用于疾病和发育模型,体外。
Introduction
人类多能干细胞 (hPSCs) 被定义为包括人类胚胎干细胞 (干细胞) 和人类诱导多能干细胞 (hiPSCs), 并具有独特的能力, 不仅在适当的生长条件下进行自我更新,而且, 可以区分为从三种细菌层派生的所有单元类型: 内胚层、胚层和外胚层1。由于这些独特的能力, hPSCs 的再生医学, 疾病建模和细胞疗法的巨大希望2。虽然多个细胞类型已成功地区分了 hPSCs, 一个重要的挑战是体外规范的完全成人样 hPSC 衍生造血干细胞 (干细胞) 和最终造血祖细胞。
一个可能的障碍, 发展的人类干细胞从 hPSCs 是存在的多个造血程序在人类胚胎的3。第一个程序出现, 称为 “原始造血”, 起源于外卵黄囊组织内, 最主要的特点是其瞬态生产的 erythroblast 祖 (EryP-氟氯化碳), 巨噬细胞, 和巨。值得注意的是, 这个程序并没有引起干细胞, 也没有引起 T 和 B 淋巴祖细胞。然而, 卵黄囊确实瞬时产生限制性的造血祖细胞, 如赤髓祖 (EMP4,5,6,7,8和红缺乏的淋巴引物多能祖 (LMPP9)。然而, 无论是 EMPs 还是 LMPPs 都是完全多能的, 或者能够在成人接受者身上植入。相比之下, 后来在发展, 经典定义的 “决定性” 的造血程序是指定的主动脉-性腺-肾区的胚胎正常, 导致所有成年造血血统, 包括 HSC。这些 intra-embryonic 明确造血细胞的规格发生在一个缺口依赖性的方式, 通过 endothelial-to-hematopoietic 过渡从 hemogenic 内皮 (他)3,10,11 ,12,13,14。除了重建能力, 这些细胞的多向电位和缺口依赖性可以用来区分这些最终的造血祖与 EMP 和 LMPP (在参考文献3,13).
了解 hPSCs 的原始和明确的造血规范的机制, 可能对在各种 hPSC 线上的最终造血祖细胞的再生生产至关重要。直到最近, hPSC 分化的协议, 可以分离多能原始和最终造血祖细胞不存在15,16,17,18, 19,20,21,22,23,24,25。许多方法使用胎牛血清 (FBS) 和/或基质共培养首先概述了 hPSC 分化的造血潜能, 与原始和最终造血电位的混合物15,16, 17,19,22,23,25。此外, 许多无血清造血协议描述的信号要求的规格胚层从 hPSCs 的海港造血电位18,20,21,24. 然而, 由于这些方法仍然产生了两种程序的异构混合物, 所以它们在临床应用和理解发展机制上的应用可能是有限的。
我们最近建立了这些研究, 概述了激活/节和 WNT 信号的阶段特定的信号要求的原始和最终的造血规范从 hPSC 派生的胚层18,26.后者是特别独特的, 因为它的使用阶段特定的 WNT 信号操作允许指定完全原始或完全明确造血祖细胞26。在胚层规范中, 标准 WNT 信号与 PORCN 抑制剂 IWP2 的抑制作用导致 CD43+ EryP-氟氯化碳和髓祖细胞的规范, 没有可检测到的淋巴潜能。与 GSK3β抑制剂 (CHIR99021) 在同一阶段的分化过程中, 对标准 WNT 信号的刺激形成鲜明对比, 导致完全缺乏可检测的 CD43+ EryP-CFC, 同时导致CD34+CD43−的规范。该种群具有髓样、 HBG表达红和 T 淋巴细胞电位。随后的分析发现, 他缺乏 CD7327、28和 CD18428的表达式, 其造血电位与缺口相关的28。此外, 单细胞克隆分析表明, 这些最终的造血谱系可以从多能单细胞28。在一起, 这些研究表明, 阶段特定的 WNT 信号操纵可以指定纯原始造血祖, 或多能缺口依赖的最终造血祖细胞。
在这里, 我们概述了我们的差异化策略, 产生完全原始或最终造血祖, 通过操纵规范 WNT 信号在胚层模式, 以及他们的下游造血谱系化验。这项协议是非常有价值的调查谁对生产的原始或最终造血祖 hPSCs 的再生医学应用。
Protocol
1. 试剂 获取单元格线; 干细胞或 hiPSCs 1 、小鼠胚胎成纤维细胞 (MEFs) 29 、OP9-DL4 基质 30 、 31 . 试剂准备 在 PBS 中准备一个0.1% 瓦特/v 的明胶溶液。4、#176 灭菌, aliquoting 后进行消毒; 准备明胶涂层 plasticware。外套 6-1.5 毫升的0.1% 明胶溶液的菜肴。室温孵育15分钟。使用…
Representative Results
在图 1中说明了从 hPSCs 中归纳出原始和最终造血祖细胞的示意图。胚层模式的规范 WNT 信号发生在 2-3 的差异, 其次是造血祖规范。 有代表性的流动细胞分析和菌落形成纤维素测定 hPSC 衍生的造血分化培养物, 如图 2所示。IWP2-treated 分化文化将产生一个独特的 CD34+CD43−</su…
Discussion
本议定书描述一个快速, 无血清, 无基质的方法分化的原始或最终造血祖细胞。胚层规范的原始或最终造血祖可以可靠地实现使用我们的协议, 这唯一的利用小分子抑制剂的规范 WNT 信号。阶段特异的 WNT 激活由 GSK3β抑制剂 CHIR9902133产生最终的造血胚层, 而 WNT 抑制剂 PORCN 抑制的 IWP234指定原始的造血胚层26。值得注意的是, 这种方法并不会产生 eng…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国华盛顿大学医学院血液科内科医学院的支持。T32HL007088 来自国家心脏、肺脏和血液研究所的支持。CMS 得到了美国血液学学者奖的支持。
Materials
| Iscove's Modified Dulbecco's Medium (IMD) | Corning | 10-016 | |
| Fetal Bovine Serum (FBS), ES cell rated | Gemini Bioproducts | 100-500 | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30396.03 | |
| L-glutamine, 200 mM solution | Life Technologies | 25030-081 | |
| Penicillin-streptomycin | Life Technologies | 15070-063 | |
| 0.25% Trypsin-EDTA | Life Technologies | 25200056 | |
| 0.05% Trypsin-EDTA | Life Technologies | 25300054 | |
| Gelatin, porcine skin, Type A | Sigma-Aldrich | G1890 | |
| Alpha-MEM | Life Technologies | 12000-022 | |
| DMEM-F12 | Corning | 10-092-CV | |
| Knock-out serum replacement | Life Technologies | 10828028 | "KOSR" |
| Non-essential amino acids (NEAA) | Life Technologies | 11140050 | |
| b-mercaptoethanol, 55 mM solution | Life Technologies | 21985023 | |
| Hydrochloric acid | Sigma-Aldrich | H1758 | |
| Fraction V, Bovine Serum Albumin | Fisher Scientific | BP1605 | |
| Ham's F12 | Corning | 10-080 | |
| N2 supplement | Life Technologies | 17502048 | |
| B27 supplement, no vitamin A | Life Technologies | 12587010 | |
| Stempro-34 (SP34) | Life Technologies | 10639011 | "SP34" |
| Growth factor reduced Matrigel | Corning | 354230 | "MAT" |
| L-absorbic acid | Sigma-Aldrich | A4403 | |
| Human serum transferrin | Sigma-Aldrich | 10652202001 | |
| Monothioglycerol (MTG) | Sigma-Aldrich | M6145 | |
| Collagenase B | Roche | 11088831001 | |
| Collagenase II | Life Technologies | 17101015 | |
| DNaseI | Calbiochem | 260913 | |
| Phosphate Buffered Saline (PBS) | Life Technologies | 14190144 | |
| bFGF | R&D Systems | 233-FB | |
| BMP4 | R&D Systems | 314-BP | |
| Activin A | R&D Systems | 338-AC | |
| VEGF | R&D Systems | 293-VE | |
| SCF | R&D Systems | 255-SC | |
| IGF-1 | R&D Systems | 291-G1 | |
| IL-3 | R&D Systems | 203-IL | |
| IL-6 | R&D Systems | 206-IL | |
| IL-7 | R&D Systems | 207-IL | |
| IL-11 | R&D Systems | 218-1L | |
| TPO | R&D Systems | 288-TP | |
| EPO | Peprotech | 100-64 | |
| Flt-3 ligand (FLT3-L) | R&D Systems | 308-FK | |
| CHIR99021 | Tocris | 4423 | |
| IWP2 | Tocris | 3533 | |
| Angiotensin II | Sigma-Aldrich | A9525 | |
| Losartan Potassium | Tocris | 3798 | |
| CD4 PerCP Cy5.5 Clone RPA-T4 | BD Biosciences | 560650 | Dilution 1:100; T cell assay |
| CD8 PE Clone RPA-T8 | BD Biosciences | 561950 | Dilution 1:10; T cell assay |
| CD34 APC Clone 8G12 | BD Biosciences | 340441 | Dilution 1:100; EHT assay |
| CD34 PE-Cy7 Clone 8G12 | BD Biosciences | 348801 | Dilution 1:100; Hemogenic endothelium |
| CD43 FITC Clone 1G10 | BD Biosciences | 555475 | Dilution 1:10; Hemogenic endothelium |
| CD45 APC-Cy7 Clone 2D1 | BD Biosciences | 557833 | Dilution 1:50; T cell assay |
| CD45 eFluor450 Clone 2D1 | BD Biosciences | 642284 | Dilution 1:50; EHT assay |
| CD56 APC Clone B159 | BD Biosciences | 555518 | Dilution 1:20; T cell assay |
| CD73 PE Clone AD2 | BD Biosciences | 550257 | Dilution 1:50; Hemogenic endothelium |
| CD184 APC Clone 12G5 | BD Biosciences | 555976 | Dilution 1:50; Hemogenic endothelium |
| 4',6-diamidino-2-phenylindole (DAPI) | BD Biosciences | 564907 | Dilution 1:10,000; T cell assay |
| OP9 DL4 cells | Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156 | ||
| MethoCult H4034 | Stemcell Technologies | 4034 | "MeC" |
| Milli-Q water purification system | EMD Millipore | ||
| 5% CO2 incubator | Set at 37 C | ||
| Multigas incubator | Set at 37 C, 5% CO2, 5% O2 | ||
| 6 well tissue culture plate | Corning | 353046 | |
| 24 well tissue culture plate | Corning | 353226 | |
| 6 well low-adherence tissue culture plate | Corning | 3471 | |
| 24 well low-adherence tissue culture plate | Corning | 3473 | |
| 35 mm tissue culture dishes | Corning | 353001 | |
| Blunt-end needle, 16 gauge | Corning | 305198 | |
| 3 cc syringes | Corning | 309657 | |
| 5 mL polypropylene test tube | Corning | 352063 | |
| 5 mL polystyrene test tube | Corning | 352058 | |
| 15 mL polypropylene conical | Corning | 430791 | |
| 50 mL polypropylene conical | Corning | 430921 | |
| 2 mL serological pipette | Corning | 357507 | |
| 5 mL serological pipette | Corning | 4487 | |
| 10 mL serological pipette | Corning | 4488 | |
| 25 mL serological pipette | Corning | 4489 | |
| Cell scrapers | Corning | 353085 | |
| 2.0 mL cryovials | Corning | 430488 | |
| 5 mL test tube with 40 µM cell strainer | Corning | 352235 | |
| 40 µM cell strainer | Corning | 352340 | |
| Cell culture centrifuge | |||
| Biosafety hood | |||
| FACS AriaII or equivalent | |||
| LSRii or equivalent | |||
| FlowJo software | TreeStar | ||
| Water bath | Set at 37 C | ||
| 0.22 µM filtration system | Corning | ||
| Autoclave | |||
| 4 C refrigerator | |||
| -20 C Freezer | |||
| -80 C Freezer |
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Cite This Article
Dege, C., Sturgeon, C. M. Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells. J. Vis. Exp. (129), e55196, doi:10.3791/55196 (2017).