ESTABLISHMENT OF AN ADHERENT CELL FEEDER LAYER FROM HUMAN UMBILICAL-CORD BLOOD FOR SUPPORT OF LONG-TERM HEMATOPOIETIC PROGENITOR-CELL GROWTH

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precur sor cells in HUCB to form a hematopoietic-supporting feeder layer in p rimary cultures. The present study shows that HUCB does contain a sign ificant accessory cell population that routinely develops into a confl uent, adherent cell layer under defined primary culture conditions. HU CB-derived adherent layers were shown to support long-term hematopoiet ic activity for an average of 4 months. This was achieved by using a c ustomized coverslip with a modified surface structure as the cell atta chment substratum and using a specialized culture feeding regime. We h ave characterized the various cell types (including fibroblasts, macro phages, and endothelial cells) and extracellular matrix proteins (incl uding fibronectin, collagen III, and laminin) that were present in abu ndance in the HUCB-derived adherent cell layer. In contrast, oil red O -staining fat cells were rarely detected. ELISA and bioassays showed t hat stem cell factor and interleukin 6 were produced by the HUCB strom al cell cultures, but interleukin 3 or granulocyte/macrophage colony-s timulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.