Colony formation of clone-sorted human hematopoietic progenitors

To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34⁺ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34⁺ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.1% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34⁺ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34⁺ cells. Approximately 15% of PB CD34⁺ cells formed colonies which erythroid bursts were predominant. CD34⁺ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34⁺, CD33⁺ cells formed GM and Mac colonies 7 to 10 times as often as CD34⁺, CD33^- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34⁺, CD13^- cells or CD34⁺, CD33^- cells. The expression of HLA-DR on CD34⁺ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34⁺ cells between BM and PB. About 10% of CD34⁺ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34⁺, CD33⁺ fraction than in CD34⁺, CD33^- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.