In the present study, we have established Dextertype 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 nineweek 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 310/10 5 cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU-Mixture, CFUgranulocyte/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.

The results obtained during this initial period are encouraging and indicate that the UCB banking and transplantation program at IMSS will help to improve already existing hematopoietic cell transplant programs in Mexico. The experience generated at IMSS may be helpful to other institutions, particularly those in developing countries.

Although the experience presented herein is still limited and the period of analysis is still short, the results obtained during these 5 years are encouraging.

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.