Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos
Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos
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DOI：
10.3791/56836
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发表时间：
2017-11-01
影响因子：
1.2
通讯作者：
Stachura, D. L.
中科院分区：
文献类型：
--
作者：
Berrun, A. C.;Stachura, D. L.
造血是一个重要的细胞过程，在这个过程中，造血干细胞和祖细胞（HSPCs）分化成多种不同的细胞系，组成成熟的血液。隔离和鉴定这些高敏感人群是困难的，因为它们是事后定义的；它们只有在分化成特定的细胞系后才能被定义。在过去的几十年里，斑马鱼（Danio rerio）已经成为研究造血的模式生物。斑马鱼胚胎在体外发育，并在受精后48小时（hpf）产生确定的HSPCs。评估HSPC分化和增殖能力的试验已经开发出来，除了用共聚焦显微镜观察专门的转基因系外，还利用移植和随后的造血系统重建。然而，对许多实验室来说，这些检测方法成本高昂、技术困难且耗时。与先前描述的方法相比，开发体外模型来评估HSPC将具有成本效益，更快，并且存在更少的困难，使实验室能够快速评估影响HSPC生物学的诱变和药物筛选。这种评估HSPC的新型体外实验是通过电镀分离的整个斑马鱼胚胎并添加仅促进HSPC分化和增殖的外源因子来完成的。胚胎分离成单个细胞，并镀上支持hspc的集落刺激因子，使它们产生由单个祖细胞产生的集落形成单位（cfu）。这些检测可以更仔细地检查负责HSPC增殖、分化和调节的分子途径，这将使研究人员能够了解脊椎动物造血及其疾病期间失调的基础。
Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.