Georgolopoulos Grigorios, Iwata Mineo, Psatha Nikoletta, Yiangou Minas, Vierstra Jeff
1Altius Institute for Biomedical Sciences, Seattle, WA 98121 USA.
2Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
J Biol Res (Thessalon). 2019 Jul 18;26:4. doi: 10.1186/s40709-019-0097-7. eCollection 2019 Dec.
Hematopoiesis is a model-system for studying cellular development and differentiation. Phenotypic and functional characterization of hematopoietic progenitors has significantly aided our understanding of the mechanisms that govern fate choice, lineage specification and maturity. Methods for progenitor isolation have historically relied on complex flow-cytometric strategies based on nested, arbitrary gates within defined panels of immunophenotypic markers. The resulted populations are then functionally assessed, although functional homogeneity or absolute linkage between function and phenotype is not always achieved, thus distorting our view on progenitor biology.
In this study, we present a protocol for unbiased phenotypic identification and functional characterization which combines index sorting and clonogenic assessment of individual progenitor cells. Single-cells are plated into custom media allowing multiple hematopoietic fates to emerge and are allowed to give rise to unilineage colonies or mixed. After colony identification, lineage potential is assigned to each progenitor and finally the indexed phenotype of the initial cell is recalled and a phenotype is assigned to each functional output.
Our approach overcomes the limitations of the current protocols expanding beyond the established cell-surface marker panels and abolishing the need for nested gating. Using this method we were able to resolve the relationships of myeloid progenitors according to the revised model of hematopoiesis, as well as identify a novel marker for erythroid progenitors. Finally, this protocol can be applied to the characterization of any progenitor cell with measurable function.
造血作用是研究细胞发育和分化的模型系统。造血祖细胞的表型和功能特征显著有助于我们理解控制命运选择、谱系特化和成熟的机制。历史上，祖细胞分离方法依赖于基于免疫表型标记物定义面板内嵌套的任意门控的复杂流式细胞术策略。然后对所得群体进行功能评估，尽管功能同质性或功能与表型之间的绝对联系并不总是能够实现，从而扭曲了我们对祖细胞生物学的看法。
在本研究中，我们提出了一种用于无偏表型鉴定和功能表征的方案，该方案结合了单个祖细胞的索引分选和克隆形成评估。将单细胞接种到定制培养基中，允许多种造血命运出现，并使其产生单谱系集落或混合集落。在集落鉴定后，将谱系潜能分配给每个祖细胞，最后召回初始细胞的索引表型，并将表型分配给每个功能输出。
我们的方法克服了当前方案的局限性，超越了既定的细胞表面标记物面板，不再需要嵌套门控。使用这种方法，我们能够根据修订后的造血模型解析髓系祖细胞的关系，并鉴定出一种新的红系祖细胞标记物。最后，该方案可应用于任何具有可测量功能的祖细胞的表征。
