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Optimization of the Cycling of Clonogenic and Primitive Cord Blood Progenitors by Various Growth Factors

Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107 - C.E.R.V.I., CHU Pitié Salpétrière, Paris, France

Key Words. Cell cycle • CD34⁺ cells • Committed progenitors • Primitive progenitors • Long-term culture • Cytokines • Cord blood

Dr. F.M. Lemoine, Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107 - C.E.R.V.I., CHU Pitié Salpétrière, 83, Bld de l'Hôpital, 75561 Paris Cedex, France.

The cycling status of cord blood progenitors and the culture conditions triggering their activation into S-phase have been studied using flow cytometry and a ³H-thymidine suicide assay. Mononuclear cells cultured either in Iscove's modified Dulbecco's medium (IMDM) ± 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medium (DMEM) ± 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34⁺ cells, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of interleukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase ~40% of CFCs within 24-48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin-like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC-IC into S-phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC-IC within two days.