Heparin sodium salt

HEK293T cells were cultured in DMEM (HyClone) supplemented with 10% FBS and 1x Penicillin Streptomycin. K562 cells were maintained in RPMI supplemented with 10% FBS and 1x Penicillin Streptomycin HUDEP2 cells were cultured in StemSpan™ SFEM II (STEMCELL Technologies) supplemented with 50 ng/ml SCF (ImmunoTools), 3 U/ml EPO (Zyrop 4000 IU Injection), 1x Pen-Strep, 1 µM dexamethasone (Alfa Aesar), 1 μg/ml doxycycline (Sigma-Aldrich) and 1x l-Glutamine 200 mM (Gibco™) (Kurita et al., 2013 (link)). Erythroid differentiation of HUDEP2 cells was carried out using two-phase erythroid differentiation media. Phase I media consisted of IMDM glutamax (Gibco), 3% AB serum (MP Biomedicals), 2% FBS, 0.1% insulin solution human (Sigma-Aldrich), 3 U/ml Heparin sodium salt (MP Biomedicals), 200 μg/ml Holo Transferrin (BBI Solutions), 3 U/ml EPO, 10 μg/ml SCF, 1 ng/ml IL3 (Immuno Tools), 1x Pen-Strep, and 1 μg/ml doxycycline. Phase II media is similar in composition except that it is devoid of doxycycline and contains 500 μg/ml of holotransferrin. Around one million cells were used to set up differentiation. Media change was done on day-3 and cells were shifted to phase II media on day-6. The culture was terminated on day-9.

For the erythroid differentiation of HUDEP-2 cells, we followed previously established protocol with slight modification (Trakarnsanga et al., 2017 (link)). After 8 days of expansion, around 1 million of edited cells were seeded in 65 mm cell culture dish (Eppendorf) with 5 ml of differentiation media consisting of IMDM glutamax (Gibco), 3% AB serum (MP Biomedicals), 2% FBS, 0.1% insulin solution human (Sigma-Aldrich), 3 U/ml Heparin sodium salt (MP Biomedicals), 200 μg/ml Holo Transferrin (BBI Solutions), 3 U/ml EPO, 10 μg/ml SCF, 1 ng/ml IL3 (Immuno Tools), 1× Pen-Strep, and 1 μg/ml doxycycline. Erythroid differentiation was carried out in 10 cm dish with regular media change (on days 3 and 6) up to the end of differentiation (for 9 days). On day 6, these cells were cultured in erythroid differentiation medium with 500 μg/ml of holotransferrin and devoid of doxycycline.
For erythroid differentiation of CD34+ cells, HSPCs were cultured in a three-phase liquid culture system and subjected to enucleation analysis as previously described (Psatha et al., 2018 (link)). The erythroid differentiation pattern was evaluated in the erythroblast obtained from HUDEP-2 cells (on day 9) and CD34+ cells (on day 21) by FACS analysis of CD235a and CD71 markers.