Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Single ventricle heart defects (SVDs) are congenital disorders that result in a variety of complications, including increased ventricular mechanical strain and mixing of oxygenated and deoxygenated blood, leading to heart failure without surgical intervention. Corrective surgery for SVDs are traditionally handled by the Fontan procedure, requiring a vascular conduit for completion. Although effective, current conduits are limited by their inability to aid in pumping blood into the pulmonary circulation. In this report, we propose an innovative and versatile design strategy for a tissue engineered pulsatile conduit (TEPC) to aid circulation through the pulmonary system by producing contractile force. Several design strategies were tested for production of a functional TEPC. Ultimately, we found that porcine extracellular matrix (ECM)-based engineered heart tissue (EHT) composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary cardiac fibroblasts (HCF) wrapped around decellularized human umbilical artery (HUA) made an efficacious basal TEPC. Importantly, the TEPCs showed effective electrical and mechanical function. Initial pressure readings from our TEPC in vitro (0.68 mmHg) displayed efficient electrical conductivity enabling them to follow electrical pacing up to a 2 Hz frequency. This work represents a proof of principle study for our current TEPC design strategy. Refinement and optimization of this promising TEPC design will lay the groundwork for testing the construct's therapeutic potential in the future. Together this work represents a progressive step toward developing an improved treatment for SVD patients. STATEMENT OF SIGNIFICANCE: Single Ventricle Cardiac defects (SVD) are a form of congenital disorder with a morbid prognosis without surgical intervention. These patients are treated through the Fontan procedure which requires vascular conduits to complete. Fontan conduits have been traditionally made from stable or biodegradable materials with no pumping activity. Here, we propose a tissue engineered pulsatile conduit (TEPC) for use in Fontan circulation to alleviate excess strain in SVD patients. In contrast to previous strategies for making a pulsatile Fontan conduit, we employ a modular design strategy that allows for the optimization of each component individually to make a standalone tissue. This work sets the foundation for an in vitro, trainable human induced pluripotent stem cell based TEPC.

Keywords: Engineered heart tissue; Fontan Conduit; Human induced pluripotent stem cells; TEPC; Tissue engineering.

Fig. 1.

Validation of hiPSCs and the evaluation of the safety of using hiPSC-CMs. (A) Schematic diagram depicting design strategy of cardiac differentiation from hiPSC. (B) Typical hiPSC colony and immunostaining with pluripotency markers (OCT4, NANOG, SSEA-4 and TRA-1–60). hiPSCs were positive for all pluripotency markers and the activity of alkaline phosphatase (AP) was observed. Nucleus was stained by DAPI. Scale bars = 100 μm. (C) Typical iPSC-CMs and positive staining for cTnT (red). Nucleus was stained by DAPI. hiPSC-CMs were negative for pluripotency markers. Scale bars = 100 μm (D) Day 12 culture were then grown in glucose-depleted DMEM media containing 4 mM lactate for 2 days to enrich cardiomyocytes. Cells (before and after enrichment) were dissociated and cytospun onto the glass slides for hiPSC-CM quantification by cTnT (green). Nucleus was stained by DAPI. Scale bars=100 μm. Samples were analyzed by Student’s T-test (*P < 0.05). (E) Karyotyping of hiPSC-CMs demonstrated stable normal chromosomal integrity. (F) Examination of teratoma formation from hiPSCs (left hind limb) and hiPSC-CMs (right hind limb) using immunodeficient mice. Scale bars = 1 cm. (G) Teratoma generated from hiPSCs injected to left hind limb. Scale bars=1 cm. (H-J) Representative H&E stained sections derived from teratoma. Arrows indicate gut-like epithelium (endoderm) (H) , cartilage (mesoderm) (I) , and neuroepithelial rosettes (ectoderm) (J). Scale bars= 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Design strategies for EHT system using hiPSC-CMs. (A) Schematic diagram depicting design strategy to produce EHTs. hiPSCs can be differentiated into beating hiPSC-CMs via temporal Wnt signaling modulation. hiPSC-CMs were then seeded into one of three tested scaffolds including degradable PGA, rat tail-derived collagen type I and decellularized porcine ECM with controllable fiber alignment (B, C and D) for the scaffold for engineering the cardiac tissue. For beating PGA, 0.7 million hiPSC-CMs were seeded onto 5 mm × 5 mm squares of PGA and cultured for two weeks. 1.2 million hiPSC-CMs were seeded into the wells in 2% agarose molds for the generation of beating cardiac rings. 1 million cells were seeded onto thin sections of decellularized porcine myocardial extracellular matrix. Scale bars = 100 μm.

Fig. 3.

Histological comparison and contractile function of EHTs generated from two types of ceil compositions including non-enriched hiPSC-CMs and enriched hiPSC-CMs with HCFs. (A) Schematic diagram depicting design strategy for EHTs. (B) Peak force, cross-sectional area and peak stress of two EHT groups including non-enriched hiPSC-CMs (non-enCMs, 1 million) as well as enriched hiPSC-CMs (0.7 million) with HCF (0.3 million) (enCMs + HCFs). Peak force and peak stress generated in EHTs with HCFs were significantly increased (Student’s T-test; *: p < 0.05, **: p < 0.01) with no change in cross-sectional area (CSA) (Student’s T-test, p = 0.42). (C) EHTs made from either non-enCMs (1 million) alone or co-culture of enCMs (0.7 million) and HCFs (0.3 million) were stained with H&E and cTnT. Nuclei were stained by DAPI. Scale bars = 100 μm.

Fig. 4.

Scaled-up large scaffold and the generation of TEPC with deceiluiarized human HUA. (A) Schematic summary of wrapping EHT on the HUA using fibroblasts as a bio-natural glue. 1.5 million fibroblasts in 400 μL of media were coated on the HUA, after pre-coating with 0.1% gelatin for 30 min, in a petridish for 1 h EHTs were manually wrapped on the HUA to produce a TEPC. TEPC was cultured in T75 flask for five days. (B) Original frame (3 × 4 mm) and scaled-up frame (15 × 14.5 mm). (C and D) 15 × 14.5 mm of frame was employed for producing large scaffolds. A total 10 million cells (7 million enriched-hiPSC-CMs and 3 million of HCFs) were seeded onto a large scaffold in the seeding bath. EHTs were statically cultured in the Teflon frames for 4 days before wrapping a vessel. (E) Decellularized HUA located on the left end of the mandrel. (F) TEPC produced with EHTs and decellularized HUA. (G) TEPC was divided into three portions and embedded in OCT and sectioned using Leica cryostat, then mounted on a glass slide. (H) TEPC sections were stained with cTnT and vimentin. Images were acquired using a Nikon 80i microscope and captured using NIS-Elements AR software. The prime symbol “’“ denotes that the image was magnified from the box in a, b and c. Here TEPC lumen was indicated by an asterisk *. Scale bar=100 μm (a,b,c) and 20 μm (a’,b’,c’).

Fig. 5.

Measurement of pressure development of TEPCs. (A) A TEPC was sutured to the tube holding chamber. Both the lumen and surrounding environment of the TEPC were filled with Tyrode solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂ , 1.8 mM CaCl₂. 0.2 mM Na₂HPO₄ , 12 mM NaHCO₃. 5.5 mM D-glucose). The luminal starting pressure was controlled by injecting Tyrode solution via microinjector (Legato 210/210P Syringe Pump) and held at 10 mmHg. A Millar catheter was inserted from right side of the TEPC and pressure was measured by an external pressure amplifier. The measurement was performed in an incubator maintained at 5% CO₂ and 37 °C. (B) Pressure generated by a TEPC (ΔP) under electrical pacing with 0, 1 and 2 Hz (100 mA current for 10 ms impulse).