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 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 approximately 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.
Movassagh
M
M
Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107-C.E.R.V.I., CHU Pitié Salpétrière, Paris, France.
Caillot
L
L
Baillou
C
C
Guigon
M
M
Lemoine
F M
FM
D018952:Antigens, CD34 / Q000032:analysis; D001798:Blood Proteins / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D013006:Growth Hormone / Q000494:pharmacology*; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects*; D006801:Humans; D016196:S Phase / Q000187:drug effects
D018952:Antigens, CD34; D001798:Blood Proteins; D006133:Growth Substances; D013006:Growth Hormone