N-ras^m-Transduced Fibroblasts Facilitate the Expansion and Engraftment of Umbilical Cord Blood Stem Cells.

Clinical trials using ex-vivo expanded umbilical cord blood (UCB) cells have thus far failed to demonstrate significant improvement in speed of hematopoietic recovery suggesting better understanding of self-renewal, proliferation, homing and engraftment capabilities of cord blood is needed. Recent progress has been made in the identification and understanding of the cellular niche for hematopoietic stem cells (HSCs) within bone marrow however attempts to identify soluble factors that regulate HSC engraftment to bone marrow have been less successful. We have demonstrated that N-ras^m transduced NIH 3T3 fibroblasts release soluble factors that support the ex-vivo expansion of UCB CD34+ hematopoietic progenitor cells (HPCs). UCB CD34+ cells were cultured in combination with cytokines : TPO, SCF, IL-6 and flt3-ligand(all at 100ng/ml). Significant (7-fold) expansion of total nucleated cells was seen on day 19 in cultures complemented with medium produced by N-ras^m -transduced fibroblasts (N-ras^m medium), compared to control cells (GFP- transduced fibroblasts (GFP medium)). Maximum expansion of UCB CD34+ cells (2-fold) and the most primitive sub-population of CD34+ CD117+ cells (2-fold) were seen on day 4 in N-ras^m medium cells compared to GFP medium cells. Furthermore, increased numbers of cycling cells were observed in UCB CD34+ cell cultures supplemented with N-ras^m medium. Significant (3-fold) expansion of CD34+CXCR4+ cells enriched with the NOD/SCID mice engrafting cells, was also observed. This increased expansion of CD34+CXCR4+ cells correlated with a 3-fold increased engraftment of human UCB cells in a NOD/SCID mouse model confirming retention of engraftment capability of expanded cells. This data demonstrates that N-ras^m -transduced fibroblasts secrete soluble factors that promote ex-vivo expansion and in-vivo engraftment of primitive human hematopoietic progenitor/stem cells derived from UCB. Proteins that are secreted by N-ras^m -transduced fibroblasts that activate hematopoiesis are currently being identified. Our data demonstrates the potential of N-ras^m-transduced fibroblasts as a reservoir for the discovery of novel secreted proteins that regulate ex-vivo expansion and engraftment of human UCB HSCs and may represent a potential novel expansion strategy allowing greater use UCB as a stem cell source for allogeneic transplantation.