Vascular endothelial growth factor (vegf)

Cardiac differentiation was conducted as described previously by our group^{12 (link)}. Briefly, SMM18 or SMMB2 cells were suspended in serum-free differentiation medium (SFD)^{7 (link)} containing Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) and F12 medium (Thermo Fisher Scientific), supplemented with B27 without retinoic acid (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), Glutamax, ascorbic acid (Wako), and 1-thioglycerol for 2 days. Then, cells were primed toward mesoderm by culturing with growth factors including activin-A (R&D Systems), BMP4 (R&D Systems), and VEGF (Wako) from day 2 to day 4. At day 4, cells were plated and induced to the cardiac lineage using bFGF (Wako), FGF10 (R&D Systems), and VEGF. After 7 days, beating-cells were observed. To enrich cardiomyocyte yields, fresh medium-containing puromycin was fed to the cells at day 7 and cultured for 3 days. Following antibiotic selection, more than 90% of cells expressing the cardiomyocyte marker, cardiac troponin T (cTNT), were obtained. The purified cardiomyocytes were used for further analyses. For the long-term culture, we plated the cardiomyocytes at day 10 and cultured in the SFD medium up to day 38 of differentiation.

Recombinant human MCP‐1 (PeproTech, Rocky Hill, NJ, USA), recombinant human IGF‐1 (Somazon; Astellas Pharma Inc., Tokyo, Japan), and recombinant human VEGF (Wako Pure Chemical Industries Ltd., Osaka, Japan) were prepared at concentrations of 1100, 1500, and 770 pg/mL to match the corresponding concentrations in MSC‐CM.19 We designated cytokine mixtures of MCP‐1, IGF‐1, and VEGF as MIV.

To perform hematopoietic differentiation, the H1 hESC line (generously provided by Prof. Tao Cheng) was co-cultured with AGM-S3 (a mouse stroma-derived cell line) as reported previously (Mao et al. 2016 ; Xu et al. 1998 (link)). This study was approved by the institutional ethics committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC). Briefly, undifferentiated H1 hESCs were seeded on irradiated AGM-S3 cells, cultured in hPSC maintenance medium (Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, F-12 nutrient mixture, 20% knockout serum replacement (KSR; Gibco), 1% L-glutamine, 1% non-essential amino acid solution (NEAA; Gibco), and 5 ng/mL basic FGF (b-FGF; Wako)) for 3 days, and then switched to hematopoiesis-inducing medium (Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum (FBS; Hyclone), 1% NEAA (Gibco), 60 ng/mL ascorbic acid (Sigma), and 20 ng/mL vascular endothelial growth factor (VEGF; Wako)) and referred to as Day 0 [D0]. The co-cultured cells were dissociated with 0.05–0.25% trypsin/EDTA (ethylenediaminetetraacetic acid) solution (Invitrogen) at the indicated times after D0 and then subjected to flow cytometry or other manipulations.

hESCs were co-cultured with mouse stromal cell line AGM-S3 to induce their differentiation into hematopoietic cells, as reported before (Chen et al., 2017 (link)). hESCs were cut into small squares of 1.5 × 10³ cells, and then plated onto the irradiated AGM-S3 cells and cultured in the hPSC maintenance medium [high glucose Dulbecco’s modified Eagle’s medium, DMEM, F-12 nutrient mixture, 20% knockout serum replacement (KSR), 1% l-glutamine, 1% non-essential amino acid solution (NEAA; Gibco), and 5 ng/ml basic FGF (b-FGF; Wako)] for 3 days (Chen et al., 2017 (link)). The medium was changed to the hematopoiesis-inducing medium (Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS; Hyclone), 1% NEAA (Gibco), 60 ng/ml ascorbic acid (Sigma), and 20 ng/ml vascular endothelial growth factor (VEGF; Wako)), referred as day 0 (D0) (Chen et al., 2017 (link); Zhou et al., 2019 (link)). The culture medium was replaced every day. The co-cultures were dissociated by 0.05–0.25% trypsin-EDTA (Invitrogen) at D8 or D14 for further analysis. This experiment was run at least three times in parallel.

Clumps of T-iPSC were transferred onto C3H10T1/2 feeder cells and cultured in Sac medium supplemented with 20 ng/ml BMP-4 (R&D) under hypoxia (5% O₂) for 6 days. Sac medium consists of IMDM (Sigma-Aldrich) supplemented with 15% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, 1% insulin–transferrin–selenium, 450 mM monothioglycerol (Nacalai), 50 µg/ml l-ascorbic acid 2-phosphate, and 50 ng/ml VEGF (Wako). The culture condition was returned to normoxia after 6 days hypoxic culture and changed to Sac medium supplemented with 30 ng/ml SCF (R&D) and 10 ng/ml Flt3L (Peprotech). On day 14, HPC were collected and transferred onto OP9DL1 cells in OP9 medium supplemented with 1% insulin–transferrin–selenium, 50 µg/ml l-ascorbic acid 2-phosphate, 1 ng/ml IL-7, and 10 ng/ml Flt3L. After 3 weeks culture on OP9DL1, DP cells underwent the maturation process.

At first, 50,000 PB-CD34⁺ cells in 2 ml of medium were plated into each well of a six-well tissue culture dish (Primaria; BD Biosciences, San Diego, CA) and cultured in a suspension manner using serum-free culture medium (StemSpam SFEM; StemCell Technologies, Vancouver, Canada) containing 50 ng/ml VEGF (Wako, Richmond, VA), 100 ng/ml stem cell factor (Wako), 20 ng/ml interleukin-6 (Wako), 100 ng/ml Flt-3 ligand (Wako), and 20 ng/ml thrombopoietin (Wako) for 7 days, as described previously.^{22 (link),35–37}