Abstract
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 li...Continue Reading
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