Physiologic Erythropoiesis with Enhanced Globin Switching and Endogenous Cytokine Production in a Long-Term Serum-free in Vitro human Bone Marrow Biomimicry

Abstract 276

Cord blood mononuclear cells (CBMNCs) are an ideal cell source for therapeutic applications although maintenance of fetal globin in expanded erythroid cells is problematic. We have previously developed a 3D bone marrow (BM) biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which expanded CBMNCs in a cytokine-free environment for at least 28 days, with or without addition of serum. Addition of near physiological concentrations (0.2U/mL) recombinant human erythropoietin (rhuEPO) to the 3D CBMNCs serum- and cytokine-free cultures at day 7 enhanced normal erythropoiesis to enucleation (from CD45⁺/CD71⁺/CD235⁻ to CD45⁻/CD71⁺/CD235⁺ stages) and promoted erythroid clonogenic capacity (CFU-E and BFU-E). However, mechanisms of erythroid expansion and globin-maturity of the cells are ill-defined. We have now extended our investigation to evaluate in situ hemoglobin and cytokine gene expression and detect cellular hemoglobin protein and cytokine production in culture supernatants. CBMNCs were separated by Ficoll-Paque density gradient centrifugation and seeded onto collagen-coated PU 3D scaffolds at 2.5×10⁶cells/scaffold (5×5×5mm³). Cultures were established with full-medium exchange every other day in 4 conditions: 2 controls (serum-free and serum-containing cultures without rhuEPO) and serum-free cultures with addition of rhuEPO at 1.845U/mL or 0.2U/mL from day 7 onwards. Culture output was evaluated by in situ analysis and by physically extracting cells from the scaffolds. β-globin (HBB) and γ-globin (HBG) genes were detected by in situ PCR from day 0 and were consistently expressed throughout the culture period in all conditions (HBB expression was consistently higher). A decline in expression of both globins was noted at days 21 and 28 only in serum-free cultures without addition of rhuEPO. Western blot analysis of extracted cells confirmed both HBB and HBG expression in the serum-containing rhuEPO-free culture. In serum-free conditions, both HBB and HBG proteins were noted only at day 7; once rhuEPO was added to these cultures, a hemoglobin switch occurred with persistence of only HBB at days 21 and 28. Even in serum-free and rhuEPO-free cultures, HBB was present, although in lower amounts than in the rhuEPO-exposed cells, which suggested that fetal globin switching and maturation towards adult erythropoiesis occurred primarily in the serum-free cultures exposed to rhuEPO. Increased expression of GM-CSF, IL-1β and TNF was observed to day 7 and declined thereafter while IL-6 expression was observed only at day 7. There was constant high expression of TGF-β throughout the culture in all conditions, whereas TPO was not detected in any condition. The erythropoietin-receptor (EPO-R) gene was detected in all conditions, yet EPO-R was shed into the supernatant mostly in the first 7 days (maximum 42pg/ml) of serum-free cultures, declining by day 14, which corresponded to the timing of rhuEPO addition. Flt-3 was consistently detected in supernatant of the serum-containing cultures, yet declined after day 14 in serum-free cultures with and without rhuEPO. Stem cell factor (SCF), critical in early stages of erythropoiesis, was not present in culture supernatants at any time-point. Interestingly, endogenous production of EPO (maximum 0.8 mU/ml) was detected by ELISA in the first 2 weeks of both serum-containing and serum-free cultures, prior to addition of rhuEPO. High EPO concentration (maximum 2600mU/ml) was still observed in the serum-free, rhuEPO-containing cultures over the entire 28 days, suggesting that EPO was not completely utilized by the maturing cells during culture and that even lower concentrations of EPO could be beneficial. EPO detection was maximal at day 21 for serum-free cultures exposed to rhuEPO; this day 21 peak, in conjunction with the known erythroid maturation kinetics within the 3D culture, the detection of endogenous EPO production at day 14, the shedding profile of EPO-R and the hemoglobin switch, suggested that the 14–21 day time-points of culture will be important for the future study of erythroid physiology within this system. In conclusion, the 3D BM biomimicry is a good model to study erythropoiesis ex vivo, using physiological concentrations of rhuEPO and serum-free conditions rendering it suitable for future clinical applications.