Single-Cell Assays Using Hematopoietic Stem and Progenitor Cells

Hematopoietic stem cells (HSCs) undergo division, making two daughter cells with unique fate decision choices, that is, whether to self-renew to maintain stemness or differentiate to committed progenitors. Since HSCs are heterogeneous in nature understanding this phenomenon at the single cell level is important. In vitro single-cell assays like the paired-daughter cell and myeloid multilineage differentiation are useful to understand this unique stem cell process. Both assays are performed using cytokine combination which allows four-lineage myeloid differentiation-neutrophil, erythroid, macrophage/monocyte, and megakaryocyte. Paired-daughter cell assay examines symmetric or asymmetric retention of four myeloid lineages after first cell division in the paired-daughter cells. Thus, it defines asymmetric versus symmetric division patterns in the paired daughter cells. Thus, this assay may provide HSC fate decision cues. Myeloid multilineage differentiation assay examines the ability of a single cell to form multipotent clones containing four or less myeloid lineages. Here, we discuss in detail methodology of these assays.