Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay.

BACKGROUND

Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out.

METHODS

Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry.

RESULTS

Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells.

CONCLUSIONS

The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures.