Establishment of an adherent cell layer from human umbilical cord blood

Abstract

In addition to bone marrow and peripheral blood, stem cells also occur in human umbilical cord blood (HUCB), and there is an increasing interest in the use of this material as an alternative source for bone marrow transplantation and gene therapy. In vitro hematopoiesis has been maintained for up to 16 weeks in HUCB cultures, but the establishment of an adherent, stromal layer has consistently failed. Adherent cell precursors among mononuclear cells from HUCB were sought for in long-term cultures. Mononuclear cells obtained from cord blood after full term, normal deliveries were cultivated at different concentrations in Iscove’s modified Dulbecco’s medium (IMDM) with weekly feeding. An adherent layer was detected in 16 of 30 cultures, 12 of which were plated at cell concentrations higher than 2 x 106 cells/ml. In contrast to bone marrow cultures, in which the stroma is detected early, in most (10/16) positive cultures from HUCB the adherent layer was identified only after the fourth week of culture. The cells never reached confluence and detached from the plate approximately four weeks after detection. May-Grünwald-Giemsa staining of positive cultures revealed fibroblast- or endothelial-like adherent cells in an arrangement different from that of bone marrow stroma in 13 samples. In two of these, the adherent cells were organized into characteristic, delimited cords of cells. Unlike bone marrow cultures, fat cells were never observed in the adherent layers. A rapid development of large myeloid cells in the first week of culture was characteristic of negative cultures and these cells were maintained for up to 12 weeks. HUCB contains adherent cell precursors which occur in lower numbers than in bone marrow and may be at a different (possibly less mature) stage of differentiation.