Factor(s) secreted by AFT024 fetal liver cells following stimulation with human cytokines are important for human LTC-IC growth

Soluble factors produced by human marrow stroma or the murine marrow derive d M2-10B4 cell line support ex vivo maintenance for 5-8 weeks of 50% of hum an long-term culture initiating cells (LTC-IC). As the AFT024 cell line sup ports LTC-IC cultured in contact conditions better than M2-10B4 feeders, we evaluated LTC-IC support in non-contact conditions above AFT024 feeders. W e show that only 15% of LTC-IC were maintained for 5 weeks in AFT024 non-co ntact cultures (n=6, P<0.05). As AFT024-conditioned media added to M2-10B4 non-contact cultures did not inhibit LTC-IC maintenance, AFT024 cells do no t secrete factors that inhibit LTC-IC growth. We next characterized heparan sulfate glycosaminoglycans (HS-GAGs) and cytokines produced by AFT024 cell s, which are both required for LTC-IC maintenance in M2-10B4 non-contact cu ltures. The size and extent of O-sulfation of HS-GAGs in AFT024 and M2-10B4 conditioned medium were similar, indicating that absence of hematopoietic specific HS-GAGs is not responsible for the lack of hematopoietic in AFT024 non-contact cultures. Levels of 13 different cytokines secreted in AFT024- and M2-10B4-conditioned medium were similar. However, addition of human SC F, G-CSF, GM-CSF, LIF, MIP-1 alpha and IL-6 in concentrations found in huma n marrow stroma-conditioned medium to AFT024 non-contact cultures increased LTC-IC-maintenance to 72% at 5 weeks. These cytokines improved LTC-IC main tenance in part through interaction with the progenitors and in part, throu gh interaction with the AFT024 feeder. Thus, although LTC-IC maintenance is poor in AFT024 non-contact cultures, addition of human cytokines enhances LTC-IC maintenance in part through indirect effects on the AFT024 feeder. C haracterization of known or novel growth factors secreted by AFT024 cells b efore and after cytokine stimulation may lead to the identification of cyto kines that support growth of human hematopoietic stem cells.