Measurement of long-term culture initiating cells (LTC-ICs) using limiting dilution: comparison of endpoints and stromal support

Abstract

Although much progress has been made in defining primitive cell phenotypes using flow cytometry and clonogenic assays, the direct study of marrow repopulating cells remains elusive. Long-term culture initiating cells (LTC-ICs) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. Two endpoints have been reported for scoring LTC-ICs in limiting dilution assays. The first endpoint described was the generation of colony forming cells (CFCs) after 5 to 8 weeks of culture. An alternative method for scoring the LTC-IC assay is to identify cobblestone area forming cells. In the present study, estimations of LTC-IC frequency were made by measuring both endpoints and by comparing LTC-IC frequencies measured using limiting dilution assays of normal human bone marrow stroma with the measurements for murine bone marrow stromal cell line M2-10B4. For assays established on normal human bone marrow stroma, there was an equivalence between the two endpoint measures. Likewise, there was an equivalence between the two types of stroma when scoring CFC generation after 5 weeks. However, a consistently higher frequency of LTC-ICs was estimated when scoring cobblestone areas compared with that found when scoring CFC generation on the M2-10B4 stroma (p < 0.0001). Although the murine bone marrow stromal cell line M2-10B4 remains a very useful and consistently reliable alternative to normal human bone marrow stroma, these data indicate that the LTC-IC populations defined by scoring cobblestone areas or by measuring the generation of CFCs on this cell line are, in contrast to those measured using bone marrow stroma, not identical.