(P 166) Expansion of Cord Blood CD34+ve Cell Fraction using Chromatin Modifying Agents, Different Supporting Stromal Cells and Different 3D Scaffolds

Cord blood provides an easily accessible source of stem cells. Transplantation is however limited by the number of cells per cord blood unit, which are insufficient for adequate haematopoietic restoration of many adults. Attempts to ameliorate this have included incomplete myeloa-blation, multiple unit transfusion and ex-vivo expansion. We present our studies in the latter. Following consent, the mononuclear cell fraction was isolated from cord blood samples by density gradient centrifugation and immediately cryopreserved. Upon thawing, samples were pooled and cultured for a week with or without DNA-modifying agents and/or supporting cells. They were then analysed by flow cytometry to assess the CD34+ cell fraction. Demethylating agent 5-aza-cytidine followed by Histone deacetylase inhibitor trichostatin A resulted in the greater increase, confirming previous studies. Co-culturing the mononuclear cells with skin-fibroblasts, cells derived from the cord stroma and from the term placenta also increased the CD34+ fraction. A further, statistically significant increase was seen upon co-culture with a mixture of all three cell-types indicating a possible synergistic effect in supporting progenitor expansion. This optimised 2-D co-culture cell mixture was then compared with the same mixture grown in 3-D scaffolds of bovine cancellous bone (Surgibone), adjacent glass beads and non-woven fabrics. Bone matrix appeared to best support CD34+ cell fraction expansion. DNA modifying agents largely obviated the effects of co-culture, possibly due to the effect of the chemical modifiers on the stromal cells themselves. Using these two sets of response modifiers in a consecutive manner may therefore produce the best effects.