定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究
Summary
在这里, 我们提出了一个简单的方法, 定量造血干细胞和祖细胞 (HSPCs) 在胚胎斑马鱼。HSPCs 从分离的斑马鱼被镀在纤维素与支持因素, 分化成成熟的血液。这使得检测血液缺陷和允许药物筛选很容易进行。
Abstract
造血是一种必需的细胞过程, 造血干细胞和祖细胞 (HSPCs) 分化成含有成熟血液的不同细胞谱系的众多。隔离和识别这些 HSPCs 很困难, 因为它们被定义为事后; 它们只能在分化成特定的细胞谱系后定义。在过去的几十年里, 斑马鱼 (斑马鱼) 已经成为研究造血的模范有机体。斑马鱼胚胎发育为宫内, 48 小时后受精 (hpf) 已经产生了明确的 HSPCs. 通过移植和随后的研究, 对 HSPC 分化和增殖能力进行了评估。重组的造血系统, 除了可视化专业的转基因线与共焦显微镜。然而, 这些化验是成本高昂, 技术上的困难, 和耗时的许多实验室。开发一个体外模型来评估 HSPCs 将是成本效益高, 速度更快, 并目前较少的困难相比, 先前所描述的方法, 允许实验室迅速评估突变和药物屏幕, 影响 HSPC 生物学。这本新的体外测定 HSPCs 的方法是通过电镀全斑马鱼胚胎和添加外源因素, 促进只有 HSPC 分化和增殖。胚胎被分离成单个细胞, 并镀有 HSPC 支持的集落刺激因子, 导致它们产生由单个祖细胞产生的菌落形成单元 (CFUs)。这些分析应允许更仔细地检查的分子通路, 负责 HSPC 增殖, 分化和调控, 这将使研究人员了解的基础脊椎动物造血和其失调在疾病期间。
Introduction
造血是使生物体存活所需的大量成熟血细胞的过程。这是一个关键的发展过程, 涉及分化的造血干细胞 (干细胞) 到各种发育限制细胞类型, 包括成熟的血液。这些干细胞必须自我更新, 使系统永远不会枯竭, 他们必须坚持从早期胚胎发育直至死亡。在脊椎动物中, 需要不断分化和增殖造血干和祖细胞 (HSPCs), 以充分补充大多数的血液细胞后有丝分裂和每天回收。干细胞先分化成限制性祖细胞的亚群, 生成成熟的血细胞;常见的淋巴祖细胞 (CLPs)1, 最终产生 T、B 和 NK 淋巴细胞, 以及常见的髓细胞祖 (中医)2 , 产生粒、红细胞、巨噬和巨。这些祖细胞致力于产生特定的细胞谱系, 并进一步分化为更发育的限制性前体细胞, 如髓红祖 (欧洲议员), 产生红细胞和血小板, 或粒细胞巨噬细胞祖 (gmp), 可产生嗜、嗜酸性粒细胞、嗜中性和巨噬细胞2。识别和隔离这些祖细胞允许识别重要的分子通路参与造血分化和许多造血疾病, 如白血病出现时, 这些前体细胞未能正确区分.
在过去的几十年中, 斑马鱼 (斑马鱼) 模型系统已经成为胚胎和成人造血研究的关键研究工具。斑马鱼是适于遗传分析, 是 phylogenetically 最低的脊椎动物模型物种, 有类似的血管和造血系统的人。斑马鱼胚胎发育前子宫,并在48小时后受精 (hpf) 生成 HSPCs3,4,5,67, 8。斑马鱼也高度肥沃, 雌性在一个单一的离合器中放置超过100鸡蛋, 允许大样本大小和实验复制。斑马鱼的胚胎是光学透明的, 允许对造血系统进行显微可视化。斑马鱼的一些荧光转基因线标记干细胞如runx1: EGFP 鱼类9、 cd41: EGFP 鱼10和kdrl:mCherry;cmyb: GFP3双阳性动物, 允许现场直播, 实时可视化 HSC 的出现和扩展在体内3,4,7,8, 9。斑马鱼的快速生成时间和发育前子宫导致其在诱变研究中的应用11,12,13,14,15和药物筛选16,17,18,19,20 , 用于对人类血液疾病有治疗前景的化合物。总的来说, 造血系统的保护, 转基因线的存在和容易的发展, 和快速的再生时间使斑马鱼成为一个廉价, 快速, 灵活, 理想的造血研究模式。
在哺乳动物的造血系统中已经开发出许多分离和检测干细胞的方法。调查人员可以利用细胞表面感受器的组合来标记干细胞21,22,23,24, 以及利用干细胞的能力来流出染料25,26。在它们被标记后, 荧光活化细胞分选 () 允许他们的物理分离。证明细胞是 HSC 需要照射宿主动物来破坏内源性 HSPCs, 移植假定的干细胞, 并观察所有成熟的血细胞类型的长期的多谱系重组。这些检测方法在小鼠中很好的工作, 因为有许多细胞表面抗体对造血细胞和近交系小鼠菌株, 促进免疫匹配的移植。然而, 很少有斑马鱼造血细胞表面抗体产生27, 阻碍了干细胞的识别和分离。最常见的方法来标记和孤立斑马鱼干细胞是与转基因动物, 藉以细胞特异启动子序列驾驶荧光蛋白的表达。研究在背主动脉腹壁上进行了可视化和枚举, 利用这一技术的显微镜干细胞3,4,8,9。其他实验室产生了斑马鱼的克隆菌株28,29 , 并在 MHC 匹配的动物中进行了成功的移植30。然而, 这些技术是成本高昂的许多实验室, 在技术上是困难的, 是费时的。为了解决这些问题, 实验室已经生成了几个体外化验, 以测试 HSPCs 的存在、增殖率和差异化能力31、32、33, 34、35、36。这些化验显示 HSPCs 的增殖和分化在体外31,32,33,34,35,36, 抢救造血缺损36, 以及一种有效的发现和检测细胞因子的方法33,34,35。它们也被用来鉴定负责 HSPC 生物学的基因31,32。在这项研究中, 我们进一步采取这些化验, 允许定量的 HSPCs 在发展中的斑马鱼胚胎。这些化验也可以用来定量的数量 HSPCs 突变动物和动物治疗的造血破坏性药物。从本质上讲, 这些化验是快速的, 目前很少的技术挑战, 是廉价的方法, 定量 HSPC 数字, 检查其增殖, 并调查块的分化。
Protocol
Representative Results
Discussion
斑马鱼模型系统已成为研究原始和最终脊椎动物造血的有效、有效和廉价的模型。快速、廉价和目前很少的技术难题的产生可以用来测试小分子, 分析突变的胚胎, 以及阐明对 HSPC 生物学重要的分子通路。体外HSPCs 从成人斑马鱼中电镀是一种研究诱变、细胞因子和造血缺损的有效方法。这项协议的基础上, 这些研究, 但利用胚胎斑马鱼。将这些化验结果应用于胚胎斑马鱼, 可以更快地收集数据…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资助由国立卫生研究院 (NIH: K01-DK087814-01A1 到 D.L.S.), 加利福尼亚州立大学生物技术教育 & 研究计划 (CSUPERB: 脊椎动物造血龛的分子控制到 D.L.S.)来自加州州立大学的研究生研究办公室 (A.C.B.)。
Materials
| 10% Bovine Serum Albumin in Iscove's MDM | StemCell Technologies | 9300 | |
| DPBS (10x) with Calcium2+ and Magnesium2+ | Life Technologies | 14080-055 | |
| HyClone PBS (1x) | GE Healthcare Life Sciences | sh30256.01 | |
| DMEM 500 mL | Corning CellGrow | 10-017-CV | |
| Ham's F12 500 mL | Corning CellGrow | 10-080-CV | |
| FBS 500 mL | Gemini Bio-Products | 100-108 | |
| HEPES 100 mL (1 M) | Gibco life technologies | 15-630-080 | |
| Penicillin/streptomycin (5000 U/mL and 5000 mg/mL) with L-Glutamine (200 mM) |
Corning Mediatech | 30-009-CI | |
| Gentamycin Sulfate 10 mL (50 mg/mL) | Corning Mediatech | 30-005-CR | |
| 1.5 mL MCF tube | FisherBrand | 05-408-129 | |
| 3 mL 23gx1 injection needle with Luer lock | BD Safety Glide | 305905 | |
| 5 mL polystyrene round bottom tube with cell strainer cap | Corning Falcon | 352235 | |
| Methocel MC | Sigma-Aldrich | 64630 | |
| 14 mL Polystyrene round bottom tube | Corning Falcon | 352057 | |
| 10 mm polystyrene easygrip Petri dish | Corning Falcon | 351008 | |
| Librease TM | Roche Sigma-Aldrich | 5401119001 | dissociation protease |
| Pronase | Roche Sigma-Aldrich | 11459643001 | dechorionation protease |
| 15 cm Petri dish | Corning Falcon | 351058 | |
| AB | Zebrafish International Resource Center (ZIRC) | ZL1 | zebrafish strain used |
| Dithiolthreitol (DTT) | Sigma-Aldrich | 646563 |
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