In vitro growth and quantification of early (CD33-/CD38-) myeloid progenitor cells: stem cell factor requirement and effects of previous chemotherapy.
OBJECTIVE
All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application. The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells.


METHODS
Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E. Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3).


RESULTS
The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture. SCF, but not IL3, was essential for that growth. The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension. An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM. In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission.


CONCLUSIONS
CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time. The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D002455:Cell Division; D002470:Cell Survival / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000032:analysis*; D063268:Sialic Acid Binding Ig-like Lectin 3; D019089:Stem Cell Factor / Q000494:pharmacology*