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 0.01% 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 in 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.
Ema
H
H
Department of Medicine, Jichi Medical School, Tochigi-ken, Japan.
Suda
T
T
Miura
Y
Y
Nakauchi
H
H
D018952:Antigens, CD34; D000943:Antigens, Differentiation / Q000276:immunology; D001773:Blood Cells / Q000166:cytology; D001853:Bone Marrow / Q000187:drug effects / Q000276:immunology; D001854:Bone Marrow Cells; D002455:Cell Division; D002469:Cell Separation / Q000379:methods; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015848:Interleukin-5 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D010641:Phenotype
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D003115:Colony-Stimulating Factors; D006133:Growth Substances; D007377:Interleukin-3; D015848:Interleukin-5; D015850:Interleukin-6; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor