FGF23 Modulates Erythroid Differentiation

BEL-A cells^{66 (link),67 (link)}, established in the lab of Deborah E. Daniels and Jan Frayne, were cultured in StemSpan™ SFEM (Stemcell Technologies) containing 50 ng/mL SCF, 3 U/mL EPO, 1 μM dexamethasone, and 1 µg/mL doxycycline. To induce erythroid differentiation, expanding cells were transferred to primary medium supplemented with 1 µg/mL doxycycline for 4 days, and for a further 4 days without doxycycline. Cells were cultured with supplementation of either 100 ng/mL FGF23 (R&D Systems) or vehicle control for 48 h before sample collection for analysis. Cell viability was determined by trypan blue exclusion test. For FACS, aliquots of 2 × 10⁵ cells were incubated with band 3 primary antibody (BRIC71; IBGRL) in PBS containing 1% (w/v) BSA (Park Scientific Ltd) and 2 mg/mL glucose (PBS-AG), followed by APC-secondary antibody (BioLegend), or with conjugate antibodies (Annexin V-FITC or CD36-Vioblue; Miltenyi Biotech). Cells were analyzed on a BD LSR Fortessa flow cytometer. From day 6 of differentiation onwards cells were incubated with 5 μg/mL Hoechst 33342 nucleic acid stain (Merck) to distinguish the erythroblast and reticulocyte populations. Propidium iodide was used to exclude non-viable cells from analyses for band 3, CD36, and percentage reticulocyte measurements. Data were analyzed using FlowJo v10.6.1 (FlowJo LLC).