OPTIMIZATION OF THE CYCLING OF CLONOGENIC AND PRIMITIVE CORD-BLOOD PROGENITORS BY VARIOUS GROWTH-FACTORS

The cycling status of cord blood progenitors and the culture condition s triggering their activation into S-phase have been studied using flo w cytometry and a H-3-thymidine suicide assay. Mononuclear cells cultu red either in Iscove's modified Dulbecco's medium (IMDM) +/- 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medi um (DMEM) +/- 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimul ated by various growth factors (GFs). Results showed that CD34(+) cell s, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord bl ood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of inter leukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase simi lar to>40% of CFCs within 24-48 h, without modifying their number exce pt in DMEM + NBS where erythroid progenitors decreased, When cells wer e stimulated in IMDM + FCS by these three GFs + insulin-like growth fa ctor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maint ained. The latter culture conditions also recruited up to 66% of LTC-I C into S-phase. Our data underline the importance of the combination o f GFs and culture media used for optimizing the cycling and maintenanc e of CFCs and LTC-IC within two days.