Cell culture

The hESCs line (HN14, 23–30 passages) was obtained from the Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research and maintained on Matrigel-coated plates with hPSCs medium mTeSR™1 (Stem Cell Technologies, CAN) and passaged every 3 days at a 1:3 ratio using 0.5 mM EDTA, and changed fresh medium every day. Cells were maintained at 37 °C and 5% CO2 [14].

OP9 stromal cells (3–15 passages) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured on 0.1% gelatinized plates in α-MEM (Gibco, USA) supplemented with 15% fetal bovine serum (FBS; Gibco, USA) until confluence. When passaged, these cells dissociated with 0.25% trypsin (Gibco) for 1 min at 37 °C. For cryopreservation, OP9 cells were suspended in 90% FBS added 10% Dimethyl Sulfoxide (Gibco) and stored in a deep freezer at -80 °C overnight, then transferred to liquid nitrogen.

OP9 cells and HN14 cells were co-cultured in a differentiation medium consisting of α-MEM medium supplemented with 10% defined fetal bovine serum (Hyclone, USA) and 100 μmol/L monothioglycerol (MTG; Sigma, USA) [9].

Different co-culture strategies

OP9 cells were randomly assigned into two groups, experimental and control groups. In the experimental group, six different seeding numbers of OP9 cells were designed for each subgroup: 3.1 × 104cells/cm2, 5.2 × 104cells/cm2, 7.3 × 104cells/cm2, 10.4 × 104cells/cm2, 13.5 × 104cells/cm2 and 16.6 × 104cells/cm2 in 6-well plates. hESCs were plated on the related stromal cell layer (0.7–1.0 × 106 cells in 6-well plate) [9]. These cells were cultured for 1 day and co-cultured with hESCs. In the control group, OP9 cells at a seeding number of 3.1 × 104cells/cm2 in 6-well plates were cultured for 4 days to reach a confluent state followed by co-culture with hESCs. Undifferentiated hESCs were harvested by treatment with 2 mg/ml dispase enzyme (Gibco, USA) and scraped to maintain the cells in small clumps. The hESCs/OP9 cocultures were incubated for up to 10 days at 37 °C in normoxic conditions and 5% CO2 with a whole-medium change on day 1 and a half-medium change on days 4, 6, 8, and 10.

Immunofluorescence staining

To determine the identity of cell line OP9, we examined the immunophenotype of OP9. They were negative for CD34 and uniformly positive for Calponin [15]. They were fixed with methanol-acetone (1:1) for 30 min at -20 °C. After washing in phosphate-buffered saline (PBS), cells were fixed with 0.05% Tween-20 and 10% goat serum in PBS for 1 h at room temperature, then incubated with primary antibodies against Calponin (Abcam, USA) and CD34 (Abcam, USA) overnight at 4 °C. Then they were washed 3 times and incubated with Donkey Anti-Rabbit IgG (H&L) (Abcam, USA) for an additional 60 min at room temperature. Finally, the cells were incubated with 4′6-diamidino-2-phenylindole (DAPI) for 20 min at room temperature.

Flow cytometry analysis

To analyze the differentiation efficiency of the coculture system, hESCs/OP9 coculture cells were treated with 0.25% trypsin (Gibco, USA) for 20 min at 37 °C on days 8, 10 and 12. Trypsinization was terminated by adding a coculture medium. Co-culture cells were harvested by centrifugation, and washed twice by washing buffer (PBS supplemented by 5% FBS) [16]. These cells were immediately incubated with APC-human TRA-1–85 (R&D, USA), PE-Cy5.5 mouse anti-human CD34 (Becton Dickinson Company, USA) for 30 min at 4 °C. After staining, these cells were washed with washing buffer, centrifuged, and resuspended with moderate washing buffer, followed by flow cytometric analysis (BD FACSAria™ II sorter (BD Biosciences, USA). Data were analyzed with FlowJo version 7.6.1 (FlowJo, LLC). For negative control, APC mouse anti-human CD34 (Becton Dickinson Company, USA) was used to HN14, while OP9 cells labeled anti-CD34 [EP373Y] (Abcam, USA) and used Donkey Anti-Rabbit IgG (H&L) (Abcam, USA) as the secondary antibody.

Magnetic sorting for CD34 positive cells

Single-cell suspension from day 10 of hESCs/OP9 co-cultures was labeled with CD34 Microbeads Kit (MACS) as recommended by the manufacturer and went through an LS separation column attached to a Midi-MACS Separator to obtain CD34+ cells. The purity of isolated CD34 + cells was always higher than 95% as evaluated by trypan blue exclusion.

RNA isolation, RT-PCR, and quantitative RT-PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA). cDNA was synthesized using 1 μg of RNA from each sample with iScript™ cDNA Synthesis Kit (Promega, USA). RT-PCR cycling conditions were as follows: initial denaturation at 95 °C for 10 min followed by 40 denaturation cycles at 95 °C for 15 s, primer annealing at 60 °C for 60 s, primer extension phase at 72 °C 15 s and final extension step at 72 °C for 7 min (Agilent Stratagene Mx3000P, Agilent Technologies, USA). GAPDH was used as an internal control. All experiments were performed three times. Sequences of primers used in this study are shown in Table 1.

Table 1 Primer sequences for RT-PCRColony-forming assay

Sorted CD34+ cells were plated at a density of 2.5 × 104 cells/dish and suspended in 1 ml methylcellulose medium (MethoCult H4434, Stem Cell Technologies) in 35 mm low adherent dishes at 37 °C in a humidified atmosphere of 5% CO2 for 14–16 days. All clonogenic progenitor assays were performed in duplicate. Colonies of more than 50 cells were scored as positive clones and defined as macrophage (M), granulocyte and macrophage (GM), granulocyte (G), erythroid (E) according to their colony morphology.

Statistical analysis

Data were analyzed with Prism 5 software (GraphPad, USA), and are presented shown as the mean ± SD. Significant differences were considered at the P < 0.05 level.