Chronological summary of the hiPSC-SMC differentiation protocols.

<p>hiPSCs and ESCs were cultured in mTeSR^TM 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.</p

hiPSC-SMC marker expression.

<p>(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).</p

Efficiency of hiPSC-SMC differentiation.

<p>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.</p

hiPSC-SMC functional assessments.

<p>(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).</p

Fluctuations in medium nutrient levels contributed to limitations in cell expansion.

<p>Glucose measurements from β-TC6 cell spheroids cultured in (A) Static cultures, and (B) stirred suspension bioreactors using high (4.5 g/L), intermediate (2.75 g/L), and low (1.0 g/L) glucose medium, as depicted by their position on the Y axis. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (C) No difference was seen comparing static to SSB cultures with any of the glucose levels. Comparison of expansion of β-TC6 spheroid cultures indicated that changing the glucose in the medium to achieve levels closer to the physiological range did not significantly improve cell expansion. (*indicates a p value of 0.027 compared with the same culture method using high glucose medium.) SSB: stirred suspension bioreactor.</p

Adjusting culture medium feed rate regulates glucose concentrations and improves cell growth compared to continuous feeding at a constant rate.

<p>(<b>A</b>) Cell counts comparing constant feed rate to adjusted feed rate from the same cultures (*represents a p value <0.05 indicating a significant difference in culture expansion). (<b>B</b>) Average culture medium glucose levels from 21 day constant feeding, and adjusted feeding bioreactor cultures. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements).</p

Continuously fed SSB eliminated glucose fluctuations and improved cell expansion.

<p>(<b>A</b>) Glucose measurements for β-TC6 spheroid culture medium using static, SSB, and CF-SSB culture methods and feeding with standard high glucose medium. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (<b>B</b>) Fold Expansion of β-TC6 spheroids over 21 days of culture comparing static, SSB, and CF culture methods. SSB: stirred suspension bioreactor. CF: Continuously fed stirred suspension bioreactor.</p

Design of continuous feed apparatus.

<p>(<b>A</b>) Schematic diagram of continuous feeding perfusion circuit with fresh medium, perfusion pump, outflow tube, and waste medium. Solid blue lines are medium connections, dashed green lines are fiber optic connections, and dotted black lines are electronic connections. (<b>B</b>) Exploded illustration of outflow tube showing small pores, which do not allow spheroids to pass through, but allow the removal of waste medium. The continuous feeding system was designed to help improve consistency of nutrient and supplement concentrations making β-TC6 cell culture parameters more stable and controlled.</p

Stirred suspension bioreactors offered no growth improvement compared to static cultures.

<p>Fold expansion of β-TC6 spheroids after twenty one days compared stirred suspension bioreactor to static culture using standard high glucose medium.</p

Continuous feeding at a constant rate cannot maintain a physiological glucose level.

<p>Culture medium glucose measurements from continuous fed cultures with high, intermediate, or low glucose medium during 21 days of expansion. The physiological glucose range is indicated by the grey bar.</p