Cd34 hspcs

G-CSF mobilized human peripheral blood CD34+ HSPCs were purchased from STEMCELL Technologies. Cells were thawed on day 0 into StemSpan serum-free expansion medium (09655, STEMCELL Technologies) supplemented with 100 ng/mL Flt3L (Peprotech), 50 ng/mL human stem cell factor (SCF; 300-07, Peprotech), 100 ng/mL TPO (Peprotech), and 1% penicillin/streptomycin (Gibco). Electroporation of RNP complexes was done on days 1 and 2 of the culture. Differentiation of CD34+ CD38 into erythroid progenitors was done in four phases of erythroid differentiation medium (EDM) consist of Iscove’s modified Dulbecco’s medium (IMDM; Gibco) supplemented with 5% human AB serum (H6914, Sigma), 10 μg/mL recombinant human insulin (I2643, Sigma), 2 units/mL heparin (H3393, Sigma), 3 units/mL Epogen (EPO, Amgen), 330 μg/mL holo-transferrin (T4132, Sigma), and 1% penicillin/streptomycin. EDM was further supplemented with 25 ng/mL human SCF and 1 ng/mL human IL-3 (Peprotech) in phase 1 of the culture (days 4–7). IL-3 was withdrawn and human SCF is decreased to 10 ng/mL in phase 2 of the culture (days 7–11). human SCF is further decreased to 2 ng/mL in phase 3 of the culture (days 12–16). human SCF was withdrawn and holo-transferrin is increased to 1 mg/mL (day 17 and beyond). Cells were collected on day 21 for flow cytometry, RNA extraction, and Giemsa stain.

Frozen human UCB CD34⁺ HSPCs were purchased from StemCell Technologies (Vancouver, BC, Canada) and used as provided (passage number 1). The CD34⁺ cell purity in the HSPCs was determined to be 98% by flow cytometry, and the viability was determined to be >97% by trypan blue staining.
CD34⁺ HSPCs were cultured in StemSpan SFEM II expansion medium (StemCell Technologies) with 100 ng/ml SCF (ProSpec, Rehovot, Israel), 100 ng/ml Flt-3L (ProSpec), 50 ng/ml TPO (ProSpec), 20 ng/ml IL-3 (ProSpec), and 20 ng/ml IL-6 (ProSpec). The initial inoculum and maintenance densities were 5 × 10⁴ and 1 × 10⁶ cells/ml, respectively. Culture media were exchanged with fresh media at one-half volume of the initial media every 3 days, and the cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.