Maintenance of hiPSCs
All experiments were carried out with the hiPSC lines DF-699T.B (WiCell) and IISH3i-CB6 (WiCell). Undifferentiated hiPSC lines were maintained on Matrigel (BD Biosciences)-coated six-well tissue culture plates in mTeSR1 serum-free medium (STEMCELL Technologies). The cells were passaged at a 1:4 split ratio every 5 days by mechanical dissociation and the medium was replaced daily.
Hematopoietic differentiation and knockdown of ER-α transcript using siRNA
For hematopoietic differentiation of hiPSCs through embryoid body (EB) formation, confluent undifferentiated hiPSCs were scraped off and transferred to Ultra-Low attachment plate (Corning). The hiPSC clumps were incubated overnight in EB differentiation medium [Knock-out-Dulbecco’s modified Eagle’s medium (KO-DMEM) supplemented with 20 % fetal bovine serum (FBS, Hyclone), 1 mM l-glutamine, 0.1 mM β-mercaptoethanol, and 1 % non-essential amino acids]. At the next day, the medium was changed with the same EB differentiation medium supplemented with hematopoietic growth factors (hGFs): 25 ng/ml bone morphogenetic protein-4 (BMP-4), 300 ng/ml stem cell factor (SCF), 10 ng/ml interleukin-3 (IL-3), 10 ng/ml IL-6, 50 ng/ml granulocyte colony stimulating factor (G-CSF), and 300 ng/ml Flt-3L. Thereafter, the medium was changed every 3 days. All hGFs were purchased from R&D systems. To investigate the effect of E2 (Sigma) on hiPSC-derived hematopoiesis, we blocked ER signals using ER antagonist 7α,β17-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) at 100 ng/ml (Tocris) in EB differentiation medium. Undifferentiated hiPSCs were transfected with ON-TARGETplusSMARTpool siRNA targeting ER-α (L-003401-00-0005, Dharmacon) for 24 h according to the manufacturer’s instructions. Non-targeting siRNA (D-001910-10-50, Dharmacon) was used as a negative control.
Flow cytometry analysis
EBs were dissociated with Collagenase IV in 37 °C and 5 % CO2 for 2 h. Single cell suspension from dissociated EBs was resuspended in 3 % FBS-PBS. The cell were passed through a 70 μm cell strainer and incubated at 4 °C for 1 h with the following fluorochrome-conjugated mouse anti-human antibodies: CD31-PE, CD34-FITC, CD45-APC, CD235a-PE, and CD71-APC (all BD Biosciences) or their corresponding isotype controls. Anti-OCT4 (BD Biosciences) and anti-ER-α (Santa Cruz) staining was identified using Alexa 488- and 647-conjugated goat anti-mouse IgG (Invitrogen). After washing with 3 % FBS-PBS, the cells were stained with 7-amino actinomycin to exclude dead cells. Flow analysis was performed on a FACSCanto II running BD FACSDiva™ (BD Biosciences) and acquired data were analyzed using FlowJo version 10 (Tree Star, Inc.).
Colony forming unit (CFU) assay
CFU assay was performed as previously described . Briefly, 10,000 cells dissociated from EBs were plated into methylcellulose H4230 (STEMCELL Technologies) supplemented with 50 ng/ml SCF, 10 ng/ml GM-CSF, 10 ng/ml IL-3, and 3 units/ml erythropoietin. Hematopoietic cell clusters were counted on the basis of morphology after incubation for 10–14 days.
Generation of hemangioblast and measurement of colony forming capability
Hemangioblasts were generated as previously described . Briefly, 2 × 105 hiPSCs were plated on Ultra-Low attachment plate in Stemline II medium (Sigma) containing BMP-4 (50 ng/ml) and VEGF (50 ng/ml) and incubated them for 48 h. Then, the half of the medium was replaced with the medium supplemented with BMP-4 (50 ng/ml), VEGF (50 ng/ml), SCF (20 ng/ml), thrombopoietin (20 ng/ml), and Flt-3L (20 ng/ml). After 3.5 days, the EBs were dissociated with 0.05 % trypsin–EDTA for 5 min and filtered through a 40 μm cell strainer. To expand hemangioblasts, the cells were resuspended in blast growth medium alone or supplementation of E2, propyl pyrazole triol (PPT, 100 nM) and diarylpropionitrile (DPN, 100 nM) and then incubated for 4–6 days. After 4–6 days, grape-like blast colonies were observed and counted in each culture condition. In order to assess colony-forming capability of blasts grown in each condition, blasts were resuspended in Stemline II medium and mixed with methylcellulose H4436 (STEMCELL Technologies). Then, cells were plated into a 12-well non-tissue culture plate. Hematopoietic cell clusters were counted on the basis of morphology after incubation for 15 days.
Harvest of hematopoietic progenitors from human umbilical cord bloods (hUCBs)
Human UCB samples were obtained from normal full-term deliveries by Caesarian section at the Kangwon National University Hospital with the Kangwon National University Hospital Institutional Review Board (KNUH-IRB)-approved (IRB approved number: KNUH-2012-11-003-008) written consent from the patients. Mononuclear cells (MNCs) were isolated using Ficoll-Paque Plue (Pharmacia Biotech) and then resuspended in 1 % FBS-PBS. Hematopoietic progenitors expressing CD34+ antigen were enriched from MNCs by negative selection using StemSep isolation system (STEMCELL Technologies) according to the manufacturer’s instructions.
For immunofluorescence staining, undifferentiated hiPSCs and hemangioblast were fixed with 4 % paraformaldehyde for 10 min and then permeabilized with 0.5 % saponin in PBS. The cells were blocked with 10 % normal goat serum (Sigma) for 30 min at room temperature. The cells were then incubated with the following primary anti-human antibodies overnight at 4 °C: OCT4 (BD Biosciences), ER-α (Santa Cruz), and CD144 (BD Biosciences). The cells were washed twice with PBS and incubated with fluorochrome-conjugated secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (Sigma) for 5 min. Fluorescent images were visualized with a fluorescence microscope (IX-71, Olympus).
Values for all measurements are presented as mean ± SD. Comparisons for all experiments were performed with Student’s t test. Significance levels were set at p < 0.05.