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.

中文翻译：

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功能独特的造血祖细胞的无偏表型鉴定

造血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表征极大地帮助了我们了解决定命运选择，血统规范和成熟度的机制。祖细胞分离的方法历来依赖于复杂的流式细胞术策略，该策略基于已定义的免疫表型标记组中嵌套的任意门。尽管并不能始终实现功能同质性或功能与表型之间的绝对联系，但仍对功能性表型进行了评估，从而扭曲了我们对祖细胞生物学的看法。在这个研究中，我们提出了无偏见的表型鉴定和功能表征的协议，该协议结合了单个祖细胞的索引排序和克隆形成评估。将单细胞接种到常规培养基中，以允许出现多种造血命运，并允许其产生单系菌落或混合菌落。在菌落鉴定后，将谱系潜能分配给每个祖细胞，最后调出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协议扩展到既定的细胞表面标记面板之外的局限性，并消除了对嵌套门控的需求。使用这种方法，我们可以根据修改后的造血模型解析骨髓祖细胞的关系，并确定红系祖细胞的新标记。最后，该协议可以应用于具有可测量功能的任何祖细胞的表征。