Swaminathan Aishwarya, Hwang Yung, Winward Ashley, Socolovsky Merav
Department of Molecular, Cell and Cancer Biology, UMASS Chan Medical School.
Department of Molecular, Cell and Cancer Biology, UMASS Chan Medical School;
J Vis Exp. 2022 Nov 4(189). doi: 10.3791/64373.
Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit CD55 CD49f CD105 CD71) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71 CD150) and P1-hi (CD71 CD150), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit CD55 CD49f CD105 CD71 CD150) contains all of the BFU-e progenitors; Population P3 (P3, Kit CD55 CD49f CD105 CD150 CD41) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit CD55 CD49f CD105 CD150 CD41) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit CD55 CD49f CD105 CD150 CD41) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.
早期红系祖细胞最初是根据其体外集落形成潜力来定义的，并被分类为爆式集落形成单位和集落形成单位，即BFU-e和CFU-e。直到最近，仍无法从新鲜分离的成年小鼠骨髓中直接前瞻性地、完整地分离出纯的BFU-e和CFU-e祖细胞。为了填补这一空白，对小鼠骨髓的单细胞RNA测序（scRNAseq）数据集进行了分析，以检测编码细胞表面标志物的基因的表达。该分析与细胞命运测定相结合，从而开发出一种新型流式细胞术方法，该方法能够识别并分离小鼠骨髓或脾脏中完整且纯的BFU-e和CFU-e祖细胞亚群。该方法还能识别其他祖细胞亚群，包括富含嗜碱性粒细胞/肥大细胞和巨核细胞潜力的亚群。该方法包括用针对Kit和CD55的抗体标记新鲜骨髓或脾脏细胞。然后将同时表达这两种标志物的祖细胞细分为五个主要群体。群体1（P1或CFU-e，Kit⁺CD55⁺CD49f⁺CD105⁺CD71⁺）包含所有CFU-e祖细胞，可进一步细分为P1-low（CD71⁻CD150⁺）和P1-hi（CD71⁺CD150⁺），分别对应早期和晚期CFU-e；群体2（P2或BFU-e，Kit⁺CD55⁺CD49f⁺CD105⁺CD71⁺CD150⁺）包含所有BFU-e祖细胞；群体P3（P3，Kit⁺CD55⁺CD49f⁺CD105⁺CD150⁺CD41⁺）富含嗜碱性粒细胞/肥大细胞祖细胞；群体4（P4，Kit⁺CD55⁺CD49f⁺CD105⁺CD150⁺CD41⁺）富含巨核细胞祖细胞；群体5（P5，Kit⁺CD55⁺CD49f⁺CD105⁺CD150⁺CD41⁺）包含具有红系、嗜碱性粒细胞/肥大细胞和巨核细胞潜力的祖细胞（EBMP）以及偏向红系/巨核细胞/嗜碱性粒细胞的多能祖细胞（MPP）。这种新方法在分析红系和其他造血祖细胞时具有更高的精确性，并且还能参考每个流式细胞术定义群体的转录组信息。
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