Growth of engineered human myocardium with mechanical loading and vascular coculture

Abstract

Rationale: The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood.

Objective: We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches.

Methods and results: Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation.

Conclusions: Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Figure 1. Rat cardiac tissue constructs organize under strain

Desmin (red) and BrdU (brown) immunostain. A) Rat neonatal cardiomyocyte (rNC) constructs conditioned under static or cyclic stress contained elongated, aligned cardiomyocytes, in comparison to constructs under no stress. B) Increased alignment was noted in static and cyclic stress groups, and co-culture of endothelial cells did not inhibit the development of cardiomyocyte alignment. Error bars = SEM; * = p<0.001 compared to rNC Only, No Stress; # = p<0.001 compared to rNC+Endo, No Stress. Inset, alignment analysis vectors depicted on micrograph of rat heart with desmin staining. C) Cardiomyocyte alignment within the constructs developed between 1 and 4 days of static stress conditioning. D) Transmission electron microscopy of cardiac constructs revealed elongated cardiomyocytes within the collagen matrix, with nuclei (nuc), numerous mitochondria (m), and contractile filaments. Higher magnification revealed mitochondria among myofibrils (myof) with scattered nascent Z-disks (arrow).

Figure 2. Characterization of ESC- and iPSC-derived human cardiac tissue constructs

A) Constructs generated from human ESC-derived cardiomyocytes stained strongly for the cardiomyocyte marker βMHC (red) and the proliferation marker BrdU (brown). High magnification (right): human cardiomyocytes were observed undergoing nuclear division within the collagen matrix. B) Constructs generated from ESC-derived cardiomyocytes subjected to static stress conditioning (lower) or no stress conditioning (upper) stained strongly for the sarcomeric protein α-actinin (green). C) Constructs generated from iPSC-derived cardiomyocytes also stained strongly for α-actinin (red). As in B, the construct edges and vector of stress conditioning are horizontal. These constructs appeared indistinguishable from the ESC-derived constructs of similar conditioning. In both cases, myofibrils appear more aligned in the static stress conditioned constructs.

Figure 3. Stress conditioning modulates human cardiomyocyte self-organization

A) hCM constructs derived from hESCs with or without endothelial cells were placed under conditions of no stress, static stress or cyclic stress and stained for βMHC (red) and BrdU (brown). Under static and cyclic stress conditions, cardiomyocytes aligned with each other in parallel to the direction of stress. B) Quantitative alignment assessment. Unstressed constructs did not have significantly different cell alignment versus 2-D cell culture. Static and cyclic stress significantly increased cell axis alignment (* = p<0.005 versus hCM only, No Stress). Co-culture of endothelial cells within the construct did not inhibit development of cardiomyocyte alignment (# = p<0.005 versus hCM+Endo, No Stress). C) By electron microscopy, human cardiac constructs with no stess conditioning contained, cardiomyocytes with numerous mitochondria (m). Nucleus, nuc. Higher magnification (below): showed relatively disorganized nascent myofibrillar bundles (myof) in the cytoplasm and scattered Z-disks (arrows) associated. D) Static stress conditioned constructs had more regular contractile filaments with interspersed Z-disks. Occasional desmosomal junctions were observed (lower right, *).

Figure 4. Stress conditioning facilitates the cell-driven development of matrix architecture

A) Assessment of collagen fiber bundle organization using picrosirius red and polarized light. Large bundle collagen fibers fluoresced yellow in the interstitium of rat myocardium and between cells in constructs generated from human ESC-derived cardiomyocytes. These fibers appeared disarrayed in no stress conditions but closely aligned under stress conditioning. B) Quantitative alignment assessment of extracellular matrix in human cardiac constructs indicated a 2-fold increase with stress conditioning. The presence or absence of human endothelium in co-culture did not affect matrix organization. * = p<0.01 versus hCM only, No Stress, # = p<0.005 versus hCM+Endo, No Stress.

Figure 5. Stress and co-culture modulate human cardiomyocyte proliferation and hypertrophy

A) Cardiomyocyte DNA synthesis was measured by βMHC and BrdU double staining in hESC-derived cardiac constructs. Data are given as fold over basal rate in the hCM-only, static stress condition. Static and cyclic stress markedly increased hESC-derived cardiomyocyte BrdU incorporation over no stress (15% and 21% increases, respectively) as did the addition of endothelial cells (19%). B) Cardiomyocyte DNA synthesis within a single experiment (n=4 per group) of iPSC-derived cardiac constructs. Co-culture and cyclic stress conditioning both significantly increase iPSC-derived cardiomyocyte DNA synthesis. C) Cardiomyocyte hypertrophy within hESC-derived cardiac constructs was assessed by βMHC immunostaining, measuring stained area within each construct, and normalizing to number of cardiomyocyte nuclei. Due to variability in input purity, data are given as fold over the hCM only, static stress condition. Cardiomyocyte area increased 2.2-fold in response to cyclic stress conditioning. D) Spontaneous beating frequency with stress conditioning in iPSC-derived cardiac constructs. The following experimental conditions were used: no stress for 4 days, 1Hz, 5% elongation cyclic stress for 1 day followed by 3 days of no stress, or 4 days under the cyclic stress condition. Afterward, beating rate was visually assessed and a time-dependent effect due to stress conditioning was observed. E) Quantitative RT-PCR was performed on iPSC-derived cardiac constructs conditioned with no or cyclic stress for 4 days to determine the mRNA transcript levels of the following contractile and hypertrophy related genes: MYH7 (βMHC), TNNT2 (cTnT), NPPA (ANP), NPPB (BNP), CACNA1C (L-type calcium channel subunit 1Cα), RYR2 (sarcoplasmic calcium channel/ryanodine receptor) and ATP2A2 (SERCA2, sarcoplasmic calcium transporter). Significance was determined by Single Factor Anova followed by Student’s t-test in comparison to the hCM only, no stress condition. * = p<0.05; ** = p<0.01; # = p<0.005 compared to the hCM Only, No Stress condition; error bars represent standard error.

Figure 6. Stromal cells affect vascular organization within the human cardiac construct

A) ESC-derived cardiac constructs were generated by co-culture with endothelial cells with and without stromal cells and immunostained for the endothelial marker CD31. Shown at high magnification, endothelial cells organized into cord networks, structures with lumens, or structures with characteristics of both (left, hCM+Endo; middle, hCM+Endo+MEF; right hCM+Endo+MSC). B) Shown at low magnification, the prevalence of endothelial structures (CD31, red, and BrdU, brown) in the constructs increased with the addition of stromal cells (left, without MSCs; right, with MSCs). C) Quantitation of endothelial structures. The total number of structures (cord and lumenal) doubled with either MSC or MEF co-culture. The number of cord structures increased by approximately 10-fold. *, p<0.05; **, p<0.01 for cord structures compared to hCM+Endo.

Figure 7. Force/length dependence in bioengineered cardiac constructs

Constructs generated from human ESC-derived cardiomyocytes were subjected to static stress conditioning for three weeks before assessment of active force development at different lengths with a force transducer and a high speed length controller. A) Steps of 4% length increase were made starting from the slack length of the bioengineered cardiac tissue construct. B) Force was continuously measured, and an increase in magnitude (insets) occurred at greater lengths. C) Passive force (baseline) recorded 15 seconds after each acute stretch was normalized to cross-sectional area, graphed against change in length, and the slope of the first 25% length change (Young’s Modulus) was determined. D) Active force twitch height at 15 seconds was graphed against construct length. The magnitude of active force increased 8-fold in a linear manner over increasing preparation lengths before leveling off at large magnitude stretches, and the slope of the first 25% length change was calculated with an R2 value of 0.9938. This Force/Length Relationship is analogous to Starling curves generated in the intact heart.

Figure 8. Cardiac engraftment

Human cardiac tissue constructs were pulsed for 1 day with BrdU and sutured onto myocardium of athymic rats. After 1 week in vivo, the hearts were excised, sectioned, and immunostained. A) βMHC and BrdU double immunostain of hESC-derived, hCM only cardiac tissue construct. Left, graft overview, right, close apposition at graft-host interface in the myocardium. B) α-actinin immunofluorescence showed sarcomeric organization of engrafted human cardiomyocytes from the hESC-derived, hCM only cardiac tissue construct. C) IPSC-derived cardiac tissue construct engrafted onto the myocardium. Top, βMHC, bottom, α-actinin, showing the graft-host interface. D) Left, close-up of the engrafted iPSC-derived cardiac construct, right, close-up of the native myocardium (red, α-actinin). E) Within the βMHC positive area of engrafted hESC-derived constructs, patent blood vessels were filled with erythrocytes (arrowheads), indicating host perfusion of the graft. F) In engrafted hESC-derived Tri-cell human cardiac tissue constructs, human CD31 immunostaining (green) was used to mark human vessels and Ter119 (red) to mark erythrocytes. Endothelial structures of human origin containing red blood cells are indicated with arrows.