Modified in vitro conditions for cord blood-derived long-term culture-initiating cells

Objective. The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. Materials and Methods. We analyzed 192 umbilical cord blood (UCB), 35 norma l bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-p rimed normal peripheral blood (NPB) samples. Standard clonogenic assays (co lony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit eryth roid [BFU-E], CPU-granulocyte erythroid megakaryocyte macrophage [GEMM]) an d standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The va riables tested were incubation temperature (37 degreesC and 33 degreesC) an d supportive stromal cell lines (NIH3T3 and M210-B4). Results. The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectiv ely). On the other hand, the BFU-E frequency was lower in cord blood sample s (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC;p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10-4). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedl y decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marr ow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/ 10(6) MNC; p < 0.04). However, when culture conditions (temperature, stroma l layers) were modified, UCB-LTC-IC frequency significantly increased, whil e the growth of early progenitors derived from adult tissues (BM and PB) di d not show any variation. Whatever culture conditions were used, the prolif erative potential of UCB LTC-IC was significantly higher with respect to bo ne marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs 5.9 +/- 5 vs 3.2 +/ - 2.2 colonies;p < 0.02 and p < 0.001 respectively). Conclusions. Optimal conditions for estimation of the LTC-IC frequency in c ord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher prolife rative potential than those from bone marrow and G-CSF-primed peripheral bl ood, their quantitation depends on the culture conditions, which makes it d ifficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necess ary to develop suitable and standardized functional assays to test UCB prog enitor content before the transplantation procedure. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.