Fig 1.
Chronological summary of the hiPSC-SMC differentiation protocols.
hiPSCs and ESCs were cultured in mTeSRTM medium on Matrigel-coated plates, with daily medium changes, until confluent (~2 days); then, differentiation into mesodermal-lineage cells was initiated on Day 0 by culturing the cells with CHIR99021 and BMP-4 in RPMI1640 medium and 2% B27. Differentiation into Synthetic SMCs or Contractile SMCs began on Day 3. Synthetic SMCs were produced by culturing the cells with VEGF-A and FGFβ in RPMI1640 medium and 2% B27 minus insulin (B27–) from Day 3 to Day 7, with VEGF-A and FGFβ in RPMI1640 and 2% B27 (with insulin) from Day 7 to Day 10, and with PDGFβ and TGFβ in RPMI1640 and 2% B27 from Day 10 to Day 14. Contractile SMCs were produced by culturing the cells with VEGF-A and FGFβ in RPMI1640 and 2% B27– from Day 3 to Day 7, and with PDGFβ and TGFβ in RPMI1640 and 2% B27 from Day 7 to Day 14. Purification was performed by maintaining the differentiated cells in 4 mM lactate RPMI1640 metabolic medium for 4 to 6 days.
Table 1.
QRT-PCR Primer Sequences.
Table 2.
Antibodies.
Fig 2.
Efficiency of hiPSC-SMC differentiation.
The efficiency of the differentiation protocols was evaluated via flow cytometry analyses of SMA expression in (A) undifferentiated cardiac-lineage hiPSCs (chiPSCs) and in (B-C) chiPSC-SMCs obtained via the (B) Synthetic and (C) Contractile SMC differentiation protocols before and after purification.
Fig 3.
(A) mRNA levels of the SMC markers alpha smooth-muscle actin 2 (αSMA-2), smooth muscle myosin heavy chain 11 (MHC-11), calponin (Calp), vascular-endothelial cadherin (VE-Cad), and transgelin (Tgln) were evaluated via quantitative RT-PCR and normalized to endogenous GAPDH mRNA levels (*p<0.01 vs Conventional or Contractile chiPSC-SMCs, †p<0.05 vs Conventional chiPSC-SMCs, †p<0.01 vs Contractile chiPSC-SMCs). (B) Smooth-muscle actin (SMA), collagen I (Col I), connexin 43 (Cnx 43), vimentin (Vmt), and calponin (Calp) protein expression (red) was detected via immunofluorescent staining in human aortic SMCs, in chiPSC-SMCs that were obtained via a conventional differentiation protocol, and in chiPSC-SMCs obtained via our Synthetic or Contractile hiPSC-SMC differentiation protocols; nuclei were counterstained with DAPI (blue) (bar = 100 μm).
Fig 4.
hiPSC-SMC functional assessments.
(A) 4×105 Synthetic or Contractile chiPSC-SMCs were cultured on gelatin-coated plates for 24 hours; then, the plate was scratched with a 200-μL pipette tip, and images of the scratched area were obtained 0 and 10 hours later. Migration was quantified by counting the number of cells that had migrated into the scratched area (*p<0.01). (B) 1×106/mL Synthetic or Contractile chiPSC-SMCs were suspended in 100 μL of RPMI1640 and cultured in the presence of PDGFβ or TGFβ for 90 min; then, the solutions were serially diluted in half six times, and cell concentrations were evaluated via optical density measurements at 490 nm (*p<0.01). (C) 2×105 Synthetic or Contractile chiPSC-SMCs were cultured on gelatin-coated plates for 24 hours; then, the cells were treated with carbachol to induce contraction, and images were obtained 0 and 5 min later. (D) Contraction was evaluated by calculating the mean cell surface area at each of the two time points (*p<0.01). (E) 1×106 Synthetic or Contractile chiPSC-SMCs were suspended in a fibrinogen gel; then, the gels were cultured with aprotinin and Rho kinase inhibitor, and the surface area of the gels was measured 0 and 3 days later (*p<0.01).