Cell lines and primary cell cultures
All cells were cultured under 5% CO 2 at 37 °C. HEK-293T and COS7 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 1% l-glutamine and 1% penicillin–streptomycin (Sigma). HUDEP-2 cells (provided by Y. Nakamura and R. Kurita) were cultured as described21.
CD34+ haematopoietic stem and progenitor cells were isolated from peripheral blood from donors and sorted using CD34+ MACS (Miltenyi) according to the manufacturer’s protocol. The cells were differentiated using a three-phase erythroid in vitro differentiation protocol as previously described52,53. In brief, the cells were expanded in StemSpan medium (Stem Cell Technologies) supplemented with 100 ng ml−1 stem cell factor (SCF) (PeproTech), 100 ng ml−1 Flt-3 (PeproTech), 100 ng ml−1 thrombopoietin (TPO) (PeproTech) and 20 ng ml−1 IL-3 (PeproTech) for a total of six days.
After six days, the cells were reseeded in phase I differentiation medium (days 1–7) consisting of Iscove’s modified Dulbecco’s medium (IMDM) (Gibco) supplemented with 2% human plasma (Stem Cell Technologies), 3% human serum (Sigma), 3 units ml−1 heparin (Sigma), 10 µg ml−1 insulin (Sigma), 3 units ml−1 erythropoietin (Janssen-Cilag), 10 ng ml−1 SCF (PeproTech), 1 ng ml−1 IL-3 (PeproTech) and 200 µg ml−1 holo-transferrin (Sigma), at a density of around 0.5 × 106–1 × 106 cells per ml for a total of seven days. Phase II medium (days 8–12) included the same cytokines except for IL-3. During phase III (days 13 and beyond) holo-transferrin was increased to 1 mg ml−1 and SCF was withdrawn. Erythroid differentiation was monitored by fluorescence-activated cell sorting (FACS) analysis and May–Grünwald–Giemsa (RAL Diagnostics) staining of cytospin slides. Images were captured from cytospin slides using a PrimoStar 3 microscope (Zeiss) coupled with an Axiocam 208 camera (Zeiss). For growth-curve analysis, cultured erythrocyte progenitor cells were seeded in the same amounts and then counted at the indicated days both by haemocytometer (Tali, Invitrogen) and by microscope with trypan blue staining (Invitrogen) to exclude dead cells. To test for TGFβ stimulation, HUDEP-2 cells were stimulated with 5 ng ml−1 TGFβ (Sigma) and sampled for SMAD3 and CCND3 mRNA expression profiling at various time points after the treatment (0 min, 60 min, 120 min, 240 min and 300 min). The inhibitory effects on this pathway were evaluated with 20 μM SB-505124 (Sigma) in 0.2% DMSO, as previously described54. All of the cells used tested negative for mycoplasma contamination.
Human blood samples
Whole-blood samples were collected from volunteers enrolled in the SardiNIA general population cohort14, at the project centre in Lanusei (Ogliastra, Sardinia). All participants gave informed consent for the study protocols, which were approved by the Sardinian Regional Ethics Committee (protocol 2171/CE). These participants have agreed in principle to be recalled for functional follow-up experiments based on their genotypes at loci of interest, such as those reported in this study. The samples collected for the experiments reported in this study were specifically selected to ensure representation of homozygous genotypes at the loci of interest, rs112233623 and rs9349205 close to the CCND3 gene. For the malaria experiments, individuals who were carriers of the most frequent allelic variants in Sardinia that have previously been associated with protection against malaria were excluded from the collection. These variants include the β039 thalassaemia mutation (rs11549407-A) in the HBB gene (frequency = 0.051), the 3.7-kb α-thalassaemia deletion (NG_000006.1:g.34164_37967del3804; frequency = 0.14) and the Mediterranean deficiency variant (rs5030868-A) in the G6PD gene (frequency = 0.083), with the exception of a small group of samples that were selected to be hemizygous for the G6PD Mediterranean deficiency variant. We also tried to ensure a balance in terms of sex in the various genotype groups being compared (Supplementary Information).
Whole blood was collected using heparin vacutainer tubes (BD Biosciences) for gene-expression and cell-cycle experiments based on the generation of erythrocyte progenitor cells, and acid–citrate–dextrose (ACD) vacutainer tubes (BD Biosciences) for P. falciparum infection experiments. The blood was kept at ambient temperature in vitro before processing for the specific experiments, all of which began on the same day. Further details about sample collection are provided in the Supplementary Information, and the overlap of donors across the functional and P. falciparum growth experiments is detailed in Supplementary Table 5.
Flow-cytometry staining
All staining steps were performed at 4 °C, with efforts made to minimize sample exposure to ambient light. For flow-cytometry analysis, 3 × 105 in-vitro-differentiated erythroid progenitor cells were washed twice in PBS and resuspended in staining buffer (1% FBS in PBS). Cells were surface-stained for 20 min with the following antibodies: 1:50 PC7-conjugated CD235a (glycophorin A, Beckman Coulter), 1:100 PE-conjugated anti-Band3 (IBGRL) and 1:20 APC-conjugated anti-CD49d (integrin α4 chain, Beckman Coulter). After washing, cells were resuspended in 500 μl FACSFlow (BD Biosciences). For the single-cell population, CD235a–PC7 versus FSC-A was used to gate on CD235a–PC7-positive cells. Erythroblast maturation was evaluated by flow cytometry through the simultaneous detection of surface markers integrin α4, glycophorin A (GPA) and Band 3, using anti-CD49d–APC, anti-CD235a–PC7 and anti-Band3–PE monoclonal antibodies, respectively. Data were collected using BD FACS Accuri C6 Plus (BD Biosciences). For cell-cycle analysis at each time point, cultured erythroid progenitor cells were collected, washed in PBS, resuspended in permeabilizing solution and incubated at 37 °C for 30 min. Propidium iodide (BD Biosciences) was then added to a final concentration of 10 μg ml−1, and cells were incubated for an additional 10 min at room temperature before analysis. Cells were acquired on a FACSCanto (BD Biosciences) coupled with FACSDiva software (v.6.1.3).
The data analysis was performed using FlowJo software (v.10.10.0).
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