Stemline 2 medium

Haematopoietic stem cells (HSCs)/CD 34⁺ cells were isolated from normal human umbilical cord blood as previously described [22 (link)]. Cord blood collected from normal full-term deliveries at Ramathibodi Hospital (ID 04-52-39) was approved by the Ethical Committee of Research on Human Beings of the Ramathibodi Hospital, Faculty of Medicine, Mahidol University. Briefly, HSCs/CD34⁺ cells from cord blood were isolated using a CD 34 isolation kit with magnetic microbead Mini-MACS columns (Miltenyi Biotech, Geramany). The purity of CD34⁺ cells after isolation was 97% as determined by flow cytometry analysis.
Growing erythroid cells (gECs), derived from HSCs/CD34⁺ cells, at a density of 5 x 10⁵ cell/well in 24-well tissue culture plates (Corning Incorporated Costar®, NY, USA) were cultured in 1 ml of complete medium containing Stemline II medium (Sigma-Aldrich Corporation, Missouri, USA) supplemented with cytokines as previously described [36 (link)]. Lysates of IE or uninfected erythrocytes (UE) from normal donor blood were added to gEC cultures, at a ratio of 1:10 (gEC:IE/UE), on day 5 and cultured at 37°C in 5% CO₂ for 24, 48, 72 h. Viable cells were determined by trypan blue dye exclusion and dead cells were stained with 2 μg/ml propidium iodide (eBioscience) and analysed by flow cytometry.

As described previously [14 ], peripheral blood mononuclear cells from Balb/c mice were vasculogenically conditioned for five days in StemLine II medium (Sigma Aldrich) containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6, all of which were obtained from PeproTech (Rocky Hill, NJ) (Fig 1B).

pCAG4 RTR2, pCAG-kGP1.1R, pCAG VSV-G, pCL20c MSCV GFP have been previously described [27 (link), 28 (link)]. pCCL-c-MNDU3 FNA10 PGK GFP which encodes FLAG tagged NUP98-HOXA10HD (FNA10) was generously provided by Keith Humphries.
Viral vectors were prepared using the previously published transient HIV lentiviral system developed at St Jude Children’s Research hospital[29 (link)], collected in Stemline II medium (Sigma-Aldrich, St. Louis, MO, Cat. No. S0192) and stored in 5 ml aliquots at -80°C. Vector preparations were titered on 293T cells in a standard manner.

PbMNCs prepared from healthy donors and patients with DM were processed in an ex vivo serum‐free expansion culture system named QQc, as described previosuly.¹⁷ For this, fresh PbMNCs (pre‐QQc) were seeded at a density of 2 × 10⁶ cells/well in six‐well Primaria plates (BD Falcon, Franklin Lakes, New Jersey) with 2 mL/well of Stemline II medium (Sigma, St. Lois, Missouri) supplemented with recombinant human vascular endothelial growth factor (rhVEGF; 50 ng/mL), rh interleukin‐6 (rhIL‐6; 20 ng/mL), rh Fms‐related tyrosine kinase‐3 ligand (rhFlt‐3L; 100 ng/mL), rh thrombopoietin (rhTPO 20 ng/mL), rh stem cell factor (rhSCF; 100 ng/mL) (all from PeproTech, Rocky Hill, New Jersey), and an antibiotic cocktail (Invitrogen), and cultured for 7 days at 37°C in a 5% CO₂ atmosphere. After 7 days, without subculture or re‐feeding, post‐QQc cells were harvested by gently pipetting and washing the wells with EDTA‐PBS, and were then suspended in an appropriate medium.

Experiments using human samples were performed with institutional approval and following guidelines from the Ethical Review Board of Juntendo University School of Medicine (No. 2017016). All volunteers provided informed consent to participate in this study. Blood was drawn (50 mL) using a heparinized syringe via forearm venipuncture of 10 healthy volunteers, aged 30–40 years (mean: 35.7 years). Peripheral blood MNCs (PBMNCs) from each donor were isolated via density-gradient centrifugation using a Lymphocyte Separation Solution (d = 1.077; Nakalai Tesque, Kyoto, Japan). A final concentration of 2 × 10⁶ PBMNCs was seeded in each well of a six-well plate (BD Falcon, Bedford, MA, USA) and cultured at 37°C and 5% CO₂ in a serum-free QQ culture for 7 days without changing media, as described previously^{10 (link)}. QQ culture consisted of serum-free Stemline II medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with an optimized growth factor/cytokine mixture of 20 ng/mL thrombopoietin, 20 ng/mL IL-6, 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, and 50 ng/mL VEGF (all from Peprotech, Rocky Hill, NJ, USA). PBMNCs cultured for 7 days in QQ culture were termed QQMNCs. Prior to QQ culture, MNCs were labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s instructions.