Bit 9500

CD3⁺ T cells were purified from BMMCs by CD3 microbeads (Miltenyi Biotec, 130-097-043) according to manufacturer’s instructions. CD4 conventional T cells (CD4⁺CD25^-) were isolated by negative selection with CD25 microbeads (Miltenyi Biotec, 130-092-983) followed by positive selection with CD4 microbeads (Miltenyi Biotec, 130-045-101). T cells were pre-stimulated with Dynabeads Human T-Activator CD3/CD28 beads (Invitrogen, 11131D) and 100 U/ml rhIL-2 in IMDM medium (Gibco, Invitrogen) containing 10% BIT 9500 (Stemcell Technologies, 09500) for 24 h. The PRDM1-overexpressing lentivirus was purchased from Sangon Biotech (Shanghai, China). After 24 h, the cells were transduced with lentiviruses and added polybrene (Sigma, USA) at a dose of 6 μg/ml, then incubated for another 24 h at 37°C and 5% CO₂. The cells were incubated for another 24 h at 37°C and 5% CO_2, and fresh medium was changed. GFP⁺ cells were isolated after a 72 h infection and cultured in IMDM containing 10% BIT 9500 with 100 U/ml rhIL-2 routinely used.

LT-HSCs were sorted into 96-well plates (one cell-one well), and then cultured for 14 days in liquid medium supplemented with 10% FBS, 20% BIT 9500 (StemCell Technologies), 2 mM ℒ-glutamine (Life Technologies), 5 × 10⁻⁵ M β-ME (Sigma-Aldrich), 10 ng/mL stem cell factor (SCF, Pepro Tech), 10 ng/mL thrombopoietin (TPO, Pepro Tech), 10 ng/mL Interleukin 13 (IL-13, Pepro Tech) and 100 U/mL penicillin/streptomycin. Three classes of colonies were defined under the microscope: large (consisting of more than 10 000 cells); intermediate (1 000-10 000 cells); and small (1-1 000 cells).

The cell line K562 (ATCC) was cultured in RPMI 1640 medium with GlutaMAX^TM (Invitrogen) supplemented with 10% fetal bovine serum at 37°C with 5% CO₂ and passaged every 3 days. CD36-positive human proerythroblasts were derived from bone marrow CD34-positive cells, which were purchased from Stem Cell Technologies (70002.1) and cultured in erythroid differentiation condition as previous described (Wong et al., 2008 (link)). Briefly, the CD34-positive cells at 10⁴ cells/ml were grown in the serum-free erythroid expansion medium containing Alpha minimum essential medium (AMEM; Mediatech) and 20% BIT9500 (Stem Cell Technologies) to achieve bovine serum albumin, recombinant human insulin and iron-saturated human transferrin at 10 mg/ml, 10 μg/ml, and 200 μg/ml, respectively. In addition, 900 ng/ml ferrous sulfate (Sigma), 90 ng/ml ferric nitrate (Sigma), 1 μM hydrocortisone (Sigma), 100 ng/ml of recombinant human stem cell factor (SCF; Stem Cell Technologies), 5 ng/ml of recombinant human interleukin-3 (IL-3; R&D Systems), and 3 IU/ml of recombinant human EPO were also included. Fresh medium was added into the culture to maintain cells at 2 × 10⁶ cells/ml. The cells were cultured for 7 days to obtain CD36-positive cells.

Erythroid progenitor cells (EPCs) were generated in vitro from peripheral blood mononuclear cells (PBMC), obtained from the leukocyte-enriched buffy coats of anonymous blood donors available for institutional research purposes from the Immunohematology and Transfusion Service, S.Orsola-Malpighi University Hospital, Bologna (authorization 0070755/1980/2014). Availability was granted under conditions complying with Italian privacy law. Neither specific ethics committee approval nor written consent from donors was required for this research project. PBMC, isolated by centrifugation in Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Milan, Italy), were cultured in IMDM (Gibco) supplemented with 20% serum substitute BIT 9500 (StemCell Technologies, Monza, Italy), and enriched with erythropoietic growth factors as described [13 (link)]. The cells were maintained at 37 °C in 5% CO₂ and used for infection experiments at day 9 ± 1, when permissiveness to B19V infection is maximal. UT7/EpoS1 cells were cultured in IMDM, supplemented with 10% FCS and 2 U/mL rhu erythropoietin, at 37 °C and 5% CO₂

Sorted EB cells were replated in methyl cellulose containing 10% plasma-derived serum (PDS, Antech; Texas), 5% PFHM II, 1× Glutamax, 1× BIT9500 (STEMCELL Technologies) and MTG (4.5 × 10–4 M), together with kit ligand (1% conditioned medium). Blast colonies were counted 4 days later^{15 (link)}.

To monitor myeloid differentiation, sorted GFP+ CD34+ cells were grown in myeloid differentiation medium X-VIVO supplemented with 20% BIT 9500 (Stem Cell Technologies), SCF 100 ng/ml, FLT3-L 10 ng/ml, IL-3 20 ng/ml, G-CSF 20 ng/ml, GM-CSF 20 ng/ml, and IL-6 20 ng/ml (PeproTech) for 4 days, the expression of the myeloid differentiation marker Mac-1 was measured by flow cytometry.

Primary MEFs were obtained from 13.5-day embryos of the indicated genotypes based on the protocol from Wicell and cultured in standard DMEM medium containing 10% FBS (Millipore) in our lab. Murine ES cell was purchased from Wicell and iPS cells were reprogramming from MEF cells with four factors (Oct4, Sox2, Klf4, and c-Myc). Murine ES and iPSC cells were cultured in ‘ES culture medium’ composed of Knock-Out DMEM (Invitrogen) supplemented with ES cell qualified FBS (20%, Millipore), mouse LIF (1000 U/ml), non-essential amino acids, L-Glutamine, and β-mercaptoethanol. Bone marrow c-kit⁺ cells were harvested from femurs of mice, enriched by CD177-streptavidin kit (Miltenyi), and cultured in standard BIT9500 (Stemcell Technologies) containing 10% FBS supplemented with murine SCF, Flt-3, and TPO before transduction.