title: A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors.
abstract: Anaemia is an important global health problem. Therefore, it is crucial to understand its pathophysiology in various genetic or infectious diseases where dyserythropoiesis is a key pathological feature. To this effect, reproducible and reliable models of erythropoiesis in vitro are much needed as investigative tools. We have developed a serum-free liquid culture model of erythropoiesis using human umbilical cord blood CD34(+) cells cultured in the cytokine combination, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) and erythropoietin (Epo), over 14 days. We found that these culture conditions favored erythroid differentiation over the expansion of the more primitive erythroid precursors. With an initiating culture density of 5x10(4) cells per ml, the nucleated cell fold expansion increased from 7.9+/-3.9 (range 4.5 to 11.1) after 4 days to 2990.2+/-1936.1 (range 626.6 to 6912.0) after 14 days in culture. Day-14 burst-forming unit-erythroid (BFU-E) frequencies peaked at day 4 (24.0+/-8.9%), with a marked decrease in BFU-E burst size as the cultures progressed. Time-course immunophenotypical profiles were characteristically erythroid with a decrease in CD34 expression (from 96.8+/-3.0% at day 0 to 0.8+/-0.8% at day 14), and a concomitant increase in the expression of erythroid-specific markers, CD36, glycophorin A (GpA) and CD71 (from 14.8+/-5.0%, 1.7+/-1.0% and 37.9+/-18.0% to 93.0+/-7.0%, 82.1+/-14.0% and 95.7+/-3.0%, respectively). Morphological studies revealed the presence of normoblasts with the complete absence of reticulocytes and mature erythrocytes after 14 days in culture. Once the culture conditions were optimized, we scaled down our culture model from 24-well plate (large-scale) to 96-well plate cultures (small-scale). We found that the small-scale cultures compared favorably with their large-scale counterpart in terms of erythroid progenitor cell proliferation and differentiation, particularly at low CD34(+) initiating cell doses. By using tumor necrosis factor-alpha (TNF-alpha), a known inhibitor of erythropoiesis, we validated our model system and showed a dose-dependent inhibition of erythroid differentiation with TNF-alpha in our cultures. Therefore, our results demonstrate a small-scale serum-free liquid culture model of erythropoiesis that is comparable with and complements our well-defined large-scale model. Our system would prove useful for screening the effects of exogenous factors on erythropoiesis in vitro.
authors.affiliation: Stem Cell and Immunotherapy, National Blood Service--Oxford, NHS Blood and Transplant, John Radcliffe Hospital, Oxford OX3 9BQ, UK.
authors.forename: Joyce O P
authors.initials: JO
authors.lastname: Cheung
authors.forename: Climent
authors.initials: C
authors.lastname: Casals-Pascual
authors.forename: David J
authors.initials: DJ
authors.lastname: Roberts
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Watt
mesh_terms: D018929:Cell Culture Techniques; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology*; D041905:Erythroid Cells / Q000166:cytology / Q000276:immunology; D004920:Erythropoiesis / Q000187:drug effects / Q000276:immunology*; D004921:Erythropoietin / Q000494:pharmacology; D005312:Fetal Blood; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008954:Models, Biological; D019089:Stem Cell Factor / Q000494:pharmacology
chemical_list: D016895:Culture Media, Serum-Free; D016207:Cytokines; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D004921:Erythropoietin; D007371:Interferon-gamma