Human ESC cultivation
The H9 hESCs (WA09) were obtained from the WiCell Research Institute (Madison, WI, USA) with the Material Transfer Agreement (No.19-W0512) and cultured on mitomycin-treated mouse embryonic fibroblasts (MEFs) from CF-1 mice in hESC culture medium (DMEM/F12 medium, 20% knock out serum replacement, 1 mM L-glutamine, 1% NEAA, 1% penicillin/streptomycin (all from Invitrogen), 0.1 mM β-Mercaptoethanol (Sigma-Aldrich) and 10 ng/mL bFGF (Peprotech). The hESCs were subcultured every 5–6 days with a treatment of collagenase IV for passaging.
Human ESC differentiation
Single-cell suspensions of H9 hESCs were obtained by treating hESCs by Gentle Cell Dissociation Reagent (GCDR) (STEMCELL), and then mesodermal EBs were generated on the ultra-low attachment 6-well plates with APEL medium (STEMCELL Technologies) supplemented with 20 ng/mL BMP4, 10 ng/mL Activin A, 25 ng/mL VEGF, 10 ng/mL SCF, 10 ng/mL bFGF (Peprotech), 3 μM CHIR99021, 4 μM SB431542 and with 10 μM Rock inhibitor (Y-27632; STEMCELL Technologies). At day 4, the culture medium was removed and fresh APEL medium containing 20 ng/mL BMP4, 50 ng/mL VEGF, and 10 ng/mL bFGF, 10 ng/mL SCF and 15 ng/mL IGF2 was replenished until day 7. The EBs at day 7 were collected and plated on Matrigel-coated wells to differentiate into HEPs in the presence of APEL differentiation medium I supplemented with 50 ng/mL IL-3, 100 ng/mL SCF, 25 ng/mL Flt3L, 50 ng/mL VEGF, 25 ng/mL IL-6, 25 ng/mL TPO, 3U/mL EPO, 10 ng/mL bFGF and 20 ng/mL IGF2. From day 11, the last step of the hematopoietic differentiation was carried out in APEL differentiation medium II supplemented with 50 ng/mL VEGF, 100 ng/mL SCF, 25 ng/mL IL-6, 25 ng/mL TPO, 25 ng/mL Flt3L, 10 ng/mL bFGF and 20 ng/mL IGF2 for 4 more days. Cultures were maintained at 37 °C under the normoxia or hypoxia (5% O2) conditions as indicated.
Analysis of mesodermal progenitor cells
hESCs and EBs at days 2 and 4 were dissociated into single-cell suspension by TrypLE (Thermo Fischer Scientific), and then cells were incubated with antibodies against human EpCAM and CD56 antibodies. Finally, DAPI (5 mg/mL) was added to gate out dead cells and flow cytometry was performed on a BD FACSCanto II (BD Biosciences) and the data were analyzed using FlowJo Software (Version 10).
Cell surface analysis
In order to perform flow cytometry analysis, hESCs and EBs at days 4 and 7, adherent and suspension cells at days 11 and 15 after the differentiation were dissociated into single-cell suspension by TrypLE (Thermo Fischer Scientific), and then cells were washed with PBS containing 0.1% FBS and stained with antibodies against human CD34, CD31, KDR, CD45 and CD144 antibodies as per manufacturer’s protocol. Dead cells were excluded from the analysis by staining with 7-AAD (Miltenyi Biotec). Finally, flow cytometry was performed on BD FACSCanto II (BD Biosciences) and the data were analyzed using FlowJo Software (Version 10). All antibodies used are listed in Additional file 1: Table S1.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using RNAiso Plus (Takara, 9109) according to the manufacturer’s manuals. RNA concentrations were determined using a UV spectrophotometer (NanoDrop One) at an absorbance ratio of 260 to 280 nm (A260/280). Reverse transcription was performed using a PCR machine (Veriti 96). All qPCR experiments were performed on a real-time PCR machine (ABI, ABI7500) with the QuantiTect SYBR Green PCR Kit (Takara) and specific primers of the genes of interest. Gene expression was quantified by the comparative cycle threshold (Ct) method. The relative amounts of target gene expression were determined by subtracting the Ct values of these genes from the Ct value of the housekeeping gene GAPDH (ΔCt). The data were presented as 2−(ΔΔCT). The primer sequences used in this study are listed in Additional file 1: Table S2.
Cell colony-forming assay
hESC-derived CD45+ cells at day 15 after differentiation and CB CD34+ cells were collected and seeded into 1 mL MethoCult H4435 (STEMCELL Technologies) for the colony forming at a frequency of 5000 cells per dish and supplemented with 10 ng/mL Flt3L, 10 ng/mL IL-6 and 50 ng/mL TPO (PeproTech). Hematopoietic colonies containing more than 50 cells were scored 14 days after the colony forming, and then the colony-forming cells (CFC) were collected for Wright–Giemsa staining.
Analysis of CFC cells
CFCs were collected and washed with PBS for three times, and then cells were incubated with the following antibodies (5μL/1 × 106 cells) against human CD45, CD235a, CD16 and CD11b. DAPI (5 mg/mL) was added to gate out dead cells. Flow cytometry was performed on a BD FACSCanto II (BD Biosciences), and the data were analyzed using FlowJo Software (Version 10). The expression of CFC-related genes was evaluated by RT-qPCR analysis with the QuantiTect SYBR Green PCR Kit (Takara) and specific primers of the genes of interest. Gene expression was quantified and presented as 2−(ΔΔCT). The primer sequences used in this study are listed in Additional file 1: Table S2.
The EBs, adherent and suspension cells were collected and fixed in 4% PFA at room temperature for 15 min. Subsequently, the cellular membranes were broken with blocking buffer that contained 0.2% Triton X-100 (Sigma-Aldrich, T8787) and nonspecific proteins were blocked with 3% normal goat serum (Jackson Immuno Research, 017–000-121) in PBS at room temperature for 45 min. Cells were incubated with primary antibodies at 4 °C overnight and next day incubated with secondary antibodies (CST, 8890S, 8889S, 4408S and 4412S). Finally, the cell nuclei were counterstained with DAPI for 10 min and imaged using CLSM (Ni-E-A1, Japan).
Isolation of human CD34
cells from umbilical cord blood
Fresh umbilical cord blood (CB) of human healthy donors was collected from Guangzhou First People's Hospital; this study was supervised and approved by the ethics committees at Guangzhou First People's Hospital and South China University of Technology (No.: K-2021-008-01). Human peripheral blood mononuclear cells (PBMC) were separated from CB by density gradient Ficoll-Histopaque isolation. Human CD34+ cells were isolated from PBMC using the EasySep human CD34 positive selection kit (STEMCELL Technologies, Cat# 18,056). DAPI−CD34+ cells were then further sorted for transplantation.
Induction of T cells in vitro
For T cell differentiation , 5 × 105 hESC-derived CD45+ cells were centrifuged with 1 × 106 OP9 cells (derived from neonatal OP/OP mouse cranium for supporting the production of T cell) to form small 3D aggregates, which were then plated onto a 0.4-mm Millicell transwell insert (EMD Millipore) placed in a six-well plate containing 1 mL of T cell differentiation medium consisting of RPMI 1640 (Gibco), 4% B27 supplement (Thermo Fisher Scientific), 30 mM l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 1% penicillin/streptomycin (Thermo Fisher Scientific), 20 ng/mL stem cell factor (SCF), 5 ng/mL FLT3L, 5 ng/mL IL-7 and 10 ng/mL DLL4 (all from PeproTech). Differentiation medium was changed every 2 days and cultured for 8 weeks. Small 3D aggregates consisting of CB CD34+ cells and OP9 cells were cultured as above described. The CD3+ TCRαβ+ cells were then analyzed by flow cytometry on the days indicated.
Mouse transplantation and analysis of engraftment
NOD-Prkdcem26Cd52Il2rgem26Cd22kitem1Cin(V831M)/Gpt immunodeficient female mice (NCG-X; 6–8 weeks old) purchased from GemPharmatech Company (Nanjing, China) were housed under sterile conditions. NCG-X mice were intravenously inoculated with 3 × 105 CB CD34+ cells or hESC-derived CD45+ cells were performed as previously described . After the indicated time, the hematopoietic reconstitution was assessed by FACS analysis of peripheral blood (PB) and bone marrow (BM) tissues. The cells harvested from PB and BM were stained with a cocktail of antibodies against CD45, CD235a, CD16, CD11b; CD45, CD56, CD19, CD3. Dead cells labeled with DAPI (Sigma) were excluded. After incubation for 30 min at 4 °C in the dark, cells were washed twice with PBS. The analysis of the cell surface phenotype was performed by BD FACSCanto II (BD Biosciences), and the data were analyzed using FlowJo Software (Version 10). All antibodies used are listed in Additional file 1: Table S1.
Generation of engineered hESCs with the EGFP and mCherry reporters
A 1.2 kb subfragment of the HOXA9 promoter (cloned from genomic DNA of LO2 cells) was inserted into a lentiviral vector harboring a mCherry reporter gene, in which the EGFP reporter gene was driven by universal EF1α promoter. hESCs were transduced with the lentiviral virus designated as EF1α-EGFP-HOXA9-mCherry construct with a multiplicity of infection (MOI) of 10 as single cells in feeder-free culture. EGFP-positive cells were then sorted and cultured on mitomycin-treated MEFs in hESC culture medium for several passages. Subsequently, the hematopoietic differentiation was recorded by living cell tracer system (JuLI Stage, Korea), and photographs were taken at the indicated time points.
The EBs at day 7 were dissociated into single cell using TrpLE (Thermo), filtered through a 70-μm filter and stained with anti-human CD34 antibody conjugated with PE, anti-human CD31 antibody conjugated with AF700 and anti-human CD45 antibody conjugated with APC/Cy7 for 30 min at 4 °C; finally, CD34+CD31+CD45−HEPs were isolated by FACS.
Immunofluorescent staining of HEPs
2 × 104 sorted hESC-derived CD34+CD31+CD45− cells and human vascular endothelial cells (HUVECs) were cultured in endothelial cell growth medium-2 (Lonza). Cells were collected and incubated with anti-VE-Cadherin antibody (1:1000, Stem Cell, 2500S) at 4 °C overnight and then incubated with secondary antibodies (CST, 4412S). Finally, cells were counterstained with DAPI (Sigma) for 10 min and imaged using CLSM (Ni-E-A1, Japan).
Vascular-like network formation assay
2 × 104 sorted hESC-derived CD34+CD31+CD45− cells and HUVECs were immediately cultured in semisolid Matrigel for tube formation assay. Inverted-phase contrast microscope (Nikon, Japan) was taken to observe the tube formation after 6 h.
Analysis for cell cycle by flow cytometry
CD45+ cells at day 15 differentiated under the normoxia and hypoxia were collected and washed twice with PBS, and then 75% precooled ethanol was added and fixed overnight for 4 °C. RNAase was dripped and incubated at 37 °C for 30 min. After washed with PBS, cells were labeled with propidium iodide (PI) for 20 min. Finally, PBS was supplemented and cell cycle was detected by flow cytometry.
Immunofluorescence staining for ROS1
CD45+ cells at day 15 differentiated under the normoxia and hypoxia were collected for the immunofluorescent staining. Cells were incubated with anti-ROS1 antibody (1:400, Bioss, bs-2504R) at 4 °C overnight and then incubated with secondary antibodies (CST, 8889S). Finally, cells were counterstained with DAPI (Sigma) for 5 min and imaged using CLSM (Ni-E-A1, Japan).
The data were subjected to statistical analysis by the Student’s t test. Results with a value of p < 0.05 were considered statistically significant.