Holotransferrin

Samples from the end of Step 1 were collected and incubated with a medium for erythroid specification and expansion. Two different recombinant factor cocktails were tested using complete StemPro34 (SP34) (Thermo Fisher Scientific) as basal media: (i) SP34 supplemented with four factors (SP34+4F): holo-transferrin (500 µg/mL; R&D Systems, Minneapolis, MN, USA), SCF (50 ng/mL), EPO (2 U/mL), and IL-3 (10 ng/m); and (ii) SP34 supplemented with six factors (SP34+6F): SCF (50 ng/mL), VEGF (50 ng/mL), IL-6 (30 ng/mL), IL-3 (30 ng/mL), thrombopoietin (TPO) (30 ng/mL/mL) (PeproTech), and EPO (3 U/mL). Both media were supplemented with 1% penicillin/streptomycin solution. SP34+4F medium was used only for cells in suspension. SP34+6F medium was adapted from the protocol of Ng et al. [34 (link)] and used for the adherent culture of EBs in 24-well plates coated with Geltrex. Under all conditions, the medium was changed every 3–4 days until D26, when the cells in the supernatant were collected for analysis.

PB MNCs were expanded for 4–10 days in a serum-free medium supplemented with a mixture of cytokines. The two main culture media (erythroid culture medium (ECM) and granulocyte culture medium (GCM)) were tested. ECM included IMDM (50%; Invitrogen) and Ham’s F12 (50%; Invitrogen) with ITS-X (100×; Invitrogen), chemically defined lipid concentrate (100×; Invitrogen), l-glutamine (100×; Invitrogen), ascorbic acid (0.05 mg/ml; Sigma), BSA (5 mg/ml; Sigma), l-thioglycerol (200 μM; Sigma), SCF (100 ng/ml; PeproTech), IL-3 (10 ng/ml; PeproTech), erythropoietin (2 U/ml; PeproTech), IGF-1 (40 ng/ml; PeproTech), dexamethasone (1 μM; Sigma), and holo-transferrin (100 μg/ml; R&D). GCM was supplemented with IMDM (50%) and Ham’s F12 (50%), ITS-X (100×), chemically defined lipid concentrate (100×), l-glutamine (100×), ascorbic acid (0.05 mg/ml), BSA (5 mg/ml), 1-thioglycerol (200 μM), Thrombopoietin (100 ng/ml; PeproTech), SCF (100 ng/ml), Flt3 ligand (100 ng/ml; PeproTech), granulocyte-colony stimulating factor (G-CSF) (100 ng/ml; PeproTech), and IL-3 (10 ng/ml). During culture, we quantified the living cells by FACS staining and counted them automatically using a cell number counting machine (Bio-Rad).

Whole blood was obtained from healthy donors (without G-CSF stimulation) and PV patients with written informed consent for research use following the Declaration of Helsinki and approved by Siriraj Institutional Review Board (SIRB) (Certificate of Approval (COA) number: SI190/2016). Peripheral CD34⁺ cells were isolated from whole blood and cultured in the three-stage erythroid culture system described previously by Griffiths et al.^{15 (link)}. Briefly, the isolated CD34⁺ cells were maintained in Iscove’s medium (Gibco) containing 3% (v/v) human AB serum (Sigma), 2% (v/v) FBS (Sigma), 10 µg/ml insulin (Sigma), 3 U/ml heparin (Sigma), 3 U/ml Epoetin-β (Roche), 10 ng/ml SCF (R&D Systems), 1 ng/ml IL-3 (R&D Systems), 200 µg/ml holo-transferrin (R&D Systems), and 1 U/ml pen/strep (Sigma). The cultured cells were counted every other day, and fresh medium was added to obtain a final concentration of 1 to 2 × 10⁵ cells/ml. The cultured cells were maintained in tissue culture flasks at 37 °C and 5% CO₂ throughout the culture period.

We expanded PB MNCs for 4–10 days in a serum-free medium supplemented with a mixture of cytokines. We used erythroid culture medium (ECM). ECM included IMDM (50%; Invitrogen) and Ham’s F12 (50%; Invitrogen) with ITS-X (100×; Invitrogen), chemically defined lipid concentrate (100×; Invitrogen), L-glutamine (100×; Invitrogen), BSA (5 mg/ml; Sigma), ascorbic acid (0.05 mg/ml; Sigma), L-thioglycerol (200 μM; Sigma), IL-3 (10 ng/ml; PeproTech), SCF (100 ng/ml; PeproTech), erythropoietin (2 U/ml; PeproTech), dexamethasone (1 μM; Sigma), IGF-1 (40 ng/ml; PeproTech), and holotransferrin (100 μg/ml; R&D).

CD34⁺ cells were isolated from a 3 ml sample of bone marrow from the proband and 50 ml peripheral blood samples from the parents and controls by positive selection using paramagnetic microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s instructions and cultured as described (Flatt et al., 2011 (link)). Briefly, cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. The base culture medium used throughout comprised IMDM (Biochrom, Germany) supplemented with 3% AB serum, 10 μg/ml insulin, 3 IU/ml heparin (all from Sigma-Aldrich, Poole, United Kingdom), 2% FCS (Hyclone, Fisher Scientific, Ltd., United Kingdom) 3 U/ml EPO (Roche, Basel, Switzerland) and 200 μg/ml holotransferrin (R&D Systems Minneapolis, MN, United States). On days 0–8 cells were cultured in base medium with 40 ng/ml SCF and 1 ng/ml IL3 (R&D systems). From days 9 to 12, in base medium with 10 ng/ml SCF and from day 13 onward in base medium with an increased holotransferrin concentration of 500 μg/ml. Cytomicrographs were prepared by cytocentrifugation (Shandon) of 2–4 × 10⁴ cells onto slides at 1350 rpm for 5 min and stained with Leishman’s (VWR) according to the manufacturer’s instructions.