Jason W. Miklas

Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes

Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca2+ handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.

Microfabricated perfusable cardiac biowire: a platform that mimics native cardiac bundle

Tissue engineering enables the generation of three-dimensional (3D) functional cardiac tissue for pre-clinical testing in vitro, which is critical for new drug development. However, current tissue engineering methods poorly recapitulate the architecture of oriented cardiac bundles with supporting capillaries. In this study, we designed a microfabricated bioreactor to generate 3D micro-tissues, termed biowires, using both primary neonatal rat cardiomyocytes and human embryonic stem cell (hESC) derived cardiomyocytes. Perfusable cardiac biowires were generated with polytetrafluoroethylene (PTFE) tubing template, and were integrated with electrical field stimulation using carbon rod electrodes. To demonstrate the feasibility of this platform for pharmaceutical testing, nitric oxide (NO) was released from perfused sodium nitroprusside (SNP) solution and diffused through the tubing. The NO treatment slowed down the spontaneous beating of cardiac biowires based on hESC derived cardiomyocytes and degraded the myofibrillar cytoskeleton of the cardiomyocytes within the biowires. The biowires were also integrated with electrical stimulation using carbon rod electrodes to further improve phenotype of cardiomyocytes, as indicated by organized contractile apparatus, higher Youngs modulus, and improved electrical properties. This microfabricated platform provides a unique opportunity to assess pharmacological effects on cardiac tissue in vitro by perfusion in a cardiac bundle model, which could provide improved physiological relevance.

Bioreactor for modulation of cardiac microtissue phenotype by combined static stretch and electrical stimulation

We describe here a bioreactor capable of applying electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.05 mm in length and 2.5 mm in width) with a pair of posts (2.78 mm in height and 0.8 mm in diameter) in each well to serve as fixation points and for measurements of contraction force. Carbon rods, stimulating electrodes, were placed into the PDMS chamber such that one pair stimulated four microwells. For feasibility studies, neonatal rat cardiomyocytes were seeded in collagen gels into the microwells. Following 3 days of gel compaction, electrical field stimulation at 3-4 V cm(-1) and 1 Hz, mechanical stimulation of 5% static strain or electromechanical stimulation (field stimulation at 3-4 V cm(-1), 1 Hz and 5% static strain) were applied for 3 days. Cardiac microtissues subjected to electromechanical stimulation exhibited elevated amplitude of contraction and improved sarcomere structure as evidenced by sarcomeric α-actinin, actin and troponin T staining compared to microtissues subjected to electrical or mechanical stimulation alone or non-stimulated controls. The expression of atrial natriuretic factor and brain natriuretic peptide was also elevated in the electromechanically stimulated group.

Hydrogel substrate stiffness and topography interact to induce contact guidance in cardiac fibroblasts.

Previous studies demonstrated the importance of substrate stiffness and topography on the phenotype of many different cell types including fibroblasts. Yet the interaction of these two physical parameters remains insufficiently characterized, in particular for cardiac fibroblasts. Most studies focusing on contact guidance use rigid patterned substrates. It is not known how the ability of cardiac fibroblasts to follow grooves and ridges changes as the substrate stiffness is decreased to match the range of stiffness found in native heart tissues. This report demonstrates a significant interactive effect of substrate stiffness and topography on cardiac fibroblast elongation and orientation using polyacrylamide substrates of different stiffness and topography.

Enrichment of live unlabelled cardiomyocytes from heterogeneous cell populations using manipulation of cell settling velocity by magnetic field.

The majority of available cardiomyocyte markers are intercellular proteins, limiting our ability to enrich live cardiomyocytes from heterogeneous cell preparations in the absence of genetic labeling. Here, we describe enrichment of live cardiomyocytes from the hearts of adult mice in a label-free microfluidic approach. The separation device consisted of a vertical column (15 mm long, 700 μm diameter), placed between permanent magnets resulting in a field strength of 1.23 T. To concentrate the field at the column wall, the column was wrapped with 69 μm diameter nickel wire. Before passing the cells through the column, the cardiomyocytes in the cell suspension had been rendered paramagnetic by treatment of the adult mouse heart cell preparation with sodium nitrite (2.5 mM) for 20 min on ice. The cell suspension was loaded into the vertical column from the top and upon settling, the non-myocytes were removed by the upward flow from the column. The cardiomyocytes were then collected from the column by applying a higher flow rate (144 μl/min). We found that by applying a separation flow rate of 4.2 μl/min in the first step, we can enrich live adult cardiomyocytes to 93% ± 2% in a label-free manner. The cardiomyocytes maintained viability immediately after separation and upon 24 h in culture.

QHREDGS Enhances Tube Formation, Metabolism and Survival of Endothelial Cells in Collagen-Chitosan Hydrogels

Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvβ3 or α5β1were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction.

Design and fabrication of biological wires.

Cardiac tissue engineering using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has facilitated the creation of in vitro diagnostic platforms to study novel small molecules and cardiac disease at the tissue level. Yet, due to the immaturity of hPSC-CMs, there is a low fidelity between tissue-engineered cardiac tissues and adult cardiac tissues. To address this challenge, we have developed a platform that combines both physical and electrical cues to guide hPSC-CMs towards a more mature state in vitro.