Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells

Abstract

Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit⁺ (K) lineage^−/lo (L) Sca-1⁺ (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1^lo. c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2⁻ HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2⁺ multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy^lo Flk-2⁻ (LT-HSCs), Thy^lo Flk-2⁺ (ST-HSCs), and Thy⁻ Flk-2⁺ multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit⁺ lin⁻ Sca-1⁺ Flk-2⁻ from many strains of mice. Fetal liver HSCs are contained within Flk-2⁻ and Flk-2⁺ KTLS cells.