Kinetics of Hematopoiesis in Dexter‐Type Long‐Term Cultures Established from Human Umbilical Cord Blood Cells

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Abstract
In the present study, we have established Dexter-type long-term cultures (D-LTC) from human umbilical cord blood (UCB) and followed the kinetics of different hematopoietic progenitor cells (HPCs)—including multipotent (colony forming unit [CFU]- Mixture), erythroid (CFU-erythroid, BFU-E), and myeloid (CFU-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage) progenitors as well as of morphologically recognizable erythroid, myeloid and lymphoid cells—during a nine-week culture period. D-LTC were also established from adult bone marrow (BM) as controls. On day 0, both UCB and BM showed similar total numbers of HPCs (about 310/105 cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU-Mixture, CFU-granulocyte/macrophage and BFU-E). A poor adherent cell layer, consisting almost exclusively of macrophages, was developed in UCB D-LTC and this correlated with a continuous decline in HPC numbers throughout the culture period. In contrast, adherent cell numbers in BM D-LTC, including fibroblasts and macrophages, were two- to fourfold higher than in UCB cultures, and the numbers of HPCs were also significantly higher, reaching plateau levels between weeks 6 and 9. In both types of cultures, erythroid and multipotent progenitors declined relatively fast, reaching undetectable levels after five weeks of culture. Myeloid progenitors, on the other hand, were sustained longer (always at higher levels in BM cultures) and were still detected by week 9. Among myeloid progenitors, a shift towards the predominance of macrophage HPCs was observed, both in UCB and BM D-LTC, and this correlated with an increase in the proportion of mature monocytes and macrophages. Taken together, our results indicate that myeloid progenitor cell growth is deficient in UCB D-LTC and suggest that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures regardless of the number and types of adherent cells present; this suggests that the stroma developed in D-LTC is not sufficient for the proliferation of these progenitor cells.