There is great potential for human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) to serve as a test bed for developmental, pharmacological, and regenerative studies. These cells can serve as therapeutic agents, which can be implanted into damage heart tissue to supplant dead cells. They can be used to assess new pharmacological treatments for heart disease. Moreover, they can be used as model systems to study the progression of developmental and pathological states of the heart. However, upon differentiation into cardiomyocytes, these cells are distinctly immature i.e. their cell size, shape, cardiac-specific markers, ploidy, nucleation, calcium handling properties, action potentials, contractility, metabolism, etc. more closely mimic that of an embryonic-stage cardiomyocyte. Therefore, in order for these cells to serve as a valid replacement or model for more developed cardiomyocytes, their structural and functional maturation must be assessed and enhanced. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. Therefore, having the ability to quantify this contraction would provide a powerful assessment tool for hPSC-CMs. Arrays of micropost have previously been employed as a means to measure the isotonic contraction of cardiomyocytes. In this work, a new micropost technique was developed in order to allow for real-time measurements of hPSC-CM contractility, to enable contractile assessment under various different culture conditions. Previous studies with immature cardiomyocytes have shown that a number of different methods are able to enhance their contractile and structural maturation. Here, hPSC-CM maturation was achieved via: i) prolonged cell culture, ii) cell alignment, iii) controlling cell-cell contact between adjacent cells, iv) altering substrate stiffness, v) electrically-stimulating the cells, and vii) treating the cells with various different biochemical agents. Assessment of hPSC-CM structural maturation was achieved by immunofluorescent analysis, while high speed imaging of micropost deflections and fluorescent calcium transients was used to quantify functional maturation. Through these studies, I found that the micropost assay is capable of accessing the contractile state of immature human cardiomyocytes, which makes it a powerful tool for developmental studies, pharmacological screening, and disease modeling applications. Furthermore, the pro-maturation environment that I developed was able to elicit cardiomyocyte maturation in the absence of any biochemical cues. Ultimately, I believe that these novel culture and analysis techniques will provide future researchers with a means to culture large populations of rapidly matured stem cell-derived cardiomyocytes, in order to effectively perform developmental, pharmacological, and therapeutic studies in a more rapid and high-throughput manner.

As cardiovascular disease remains to be the leading cause of death worldwide, cardiac regenerative medicine aims to apply design methods to develop functional cardiac tissue for directed therapy as well as in vitro screening assays. Research in this area has shown varying degrees of success, but fully functional cardiac tissue remains to be achieved. This short-coming is due to failures in mimicking native heart tissue in vitro. The extracellular matrix (ECM) of the heart is a complex structure responsible for both biochemical and mechanical cues to the surrounding myocardium. Past research has relied heavily on the use of native biochemical signals of the ECM to influence cardiomyocyte function, but the mechanical signals of heart ECM have been less studied. The ECM of the heart is made up of aligned collagen fibers as well as other important proteins in the basement membrane responsible for cell-cell and cell-ECM interactions. The nanoscale collagen fibers have been shown to play a major role in the structural architecture of the overlying macroscopic myocardium. Advancements in nanofabrication techniques have made it possible to study the effect of substrate nanotopography on cardiomyocyte structure and function. The proteins of the basement membrane including laminin and fibronectin have been shown to strongly influence the adhesion of cardiomyocytes through integrin interactions. Recently, a specific repeating amino acid sequence, Arg-Gly-Asp (RGD), found in many native adhesion proteins, has been shown to promote cell adhesion in vitro1[superscript comma]2. Here we present a platform in which we are able to study the effect of nanoscale structural cues as well as ECM biochemical signals on maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using a customized 4 x 4 island nanopatterned substrate, nanogroove widths ranging from 350nm to 2000nm were investigated. We also present the synthesis and incorporation of bifunctionalized peptide, PUA binding peptide-RGD (PUABP-RGD) into the platform to further study the effect of native ECM-like biochemical cues on the structural maturation of hPSC-CMs.

Cardiac tissue engineering enables the generation of functional human cardiac tissue using cells in combination with biocompatible materials. Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hPSC-derived cardiac tissues. In the first part of the study, cardiomyocytes derived from human induced pluripotent stem cells (hIPSCs) were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subject to different stress and electrical pacing conditions. This engineered human myocardium exhibits Frank-Starling curve-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated at least 10-fold increase in contractility and tensile stiffness, greater cell alignment, and a 1.5-fold increase in cell size and cell volume fraction within the constructs. Stress conditioning also increased sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA2) expression. When electrical pacing was combined with static stress conditioning, the tissues showed an additional 2-fold increase in force production, tensile stiffness, and contractility, with no change in cell alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. These studies demonstrate that electrical pacing and mechanical stimulation promote both the structural and functional maturation of hiPSC-derived cardiac tissues. In the second part of the study, cardiovascular progenitor (CVP) cells derived from hPSC were used as the input cell population to generate engineered tissues. The effects of a 3-D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue were evaluated. Compared to 2-D culture, the unstressed 3-D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3-D culture suppressed smooth muscle cell maturation. Mechanical stress conditioning further improved cardiomyocyte maturation. Cyclic stress-conditioning increased expression of several cardiac markers, like beta-myosin and cTnT, and the tissue showed enhanced force production. This 3-D system has facilitated understanding of the effect of mechanical stress on the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissues. In conclusion, we were able to create a complex engineered human cardiac tissue with both stem cell-derived cardiomyocytes and CVP cells. We showed that how environmental stimulations like mechanical stress, electrical pacing, and 3-D culturing can affect the maturation and specification of cells within the engineered cardiac tissues. The study paves our way to further apply these engineered cardiac tissues to other in vitro and in vivo usages like drug testing, clinical translation, and disease modeling.

This dissertation, "Facilitated Maturation of Cardiomyocytes Derived From Human Embryonic Stem Cells in 3D Collagen Matrix Upon Mesenchymal Cell Supplementation and Mechanical Stretch" by Wei, Alvin, Zhang, 張偉, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as promising cell source for regenerative medicine, drug testing and disease modeling. Nevertheless, these cardiomyocytes are immature in terms of contractile structure, metabolism and electrophysiological properties. There are increasing efforts using biological, chemical and physical approaches to facilitate maturation of hESC-CMs, with 3D matrix recognized as an optimal in vitro platform. In light of the previous findings, cardiac tissue strips were fabricated by encapsulating hESC-CMs into collagen/matrigel matrix in current study. The engineered tissue strips contract against mounted ends and grow into compact tissues with spontaneous beating. We hypothesize that addition of mesenchymal cells in small amount could accelerate maturation of hESC-CMs in collagen matrix, with mechanical stretch assumed to be superior to static stress in driving hESC-CM maturation. More specifically, we aim to demonstrate functional improvements of engineered cardiac tissue strips in terms of structural arrangement, mechanical properties, contractile performance and gene expression. Results showed that supplementation of mesenchymal cells at 3% could already boost maturation of fabricated heart tissue strips, where benefits of mesenchymal stem cell addition were shown to be comparable to that of fibroblast. Both cell types significantly promoted compaction and cell spreading to the same extent, with similar molecular signature in terms of gene expression and protein localization shown at tissue level. hMSC co-encapsulated tissues possess greater mechanical properties than hFB added counterparts such as elastic modulus, passive tension and twitch force under strain, yet the difference was not significant. Cyclic stretch was demonstrated to render better maturated engineered cardiac tissues when comparing with static stress, with static stretch showed similar advantages, albeit to a lesser extent. Both stretch schemes outperformed static stressed samples, as evidenced by more elongated sarcomere, stronger twitch force, steeper stress-strain curve, greater elastic modulus and better expression of major contractile and hypertrophic genes. However, statistical significance was achieved only between cyclic stretched tissue strips and static stressed group in most of the evaluation assays, suggesting superiority of the cyclic stretch in functionalizing engineered cardiac tissue. In vitro maturation of cardiomyocytes is a complex process, which could be achieved through combination of multiple approaches such as mechanical loading, electrical stimulation, niche cell addition and perfusion. This study proved that mesenchymal stem cells could be considered equivalent to fibroblasts in facilitating maturation of hESC-CMs within 3D collagen matrix. Moreover, mode of loading does affect functionality of engineered cardiac tissue, with cyclic stretch demonstrated to elicit greatest improvement. Findings of current study contribute to bioengineering of functional heart tissue from hESC-derived cardiomyocytes in the long run. DOI: 10.5353/th_b5689289 Subjects: Heart cells Embryonic stem cells

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as exciting new tools in the field of cardiac research that possess immense therapeutic potential and can serve as innovative pre-clinical platforms for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Most prior efforts on improving hPSC-CM maturation have utilized cues stemming from the in vivo cardiac milieu. While these studies have resulted in modest hPSC-CM maturation improvements, individually they fail to fully recapitulate the adult cardiomyocyte phenotype. Thus, the field has gravitated towards the creation of more biomimetic microenvironments that have the ability to combine multiple signaling factors into one in vitro culture platform. Herein we provide a 2D in vitro substrate platform inspired by the myocardial microenvironment that improves hPSC-CM maturation while simultaneously allowing for quantitative measurements of mechanical and electrophysiological outputs. Substrate stiffness and micropatterned ECM are two known hPSC-CM pro-maturation cues. We study the effects of these parameters on hPSC-CMs and cardiac fibroblast generated from human induced pluripotent stem cells (hiPSC-CFs). Similar to our prior result on glass substrates, hPSC-CMs patterned on 10kPa PDMS align their internal cytoskeletal network in accordance with the micropatterned lanes. We mimic a functional cardiac syncytium by connecting micropatterned lanes with micropatterned bridges seeded with hPSC-CMs. hPSC-CMs patterned on this 15 degree chevron pattern displays anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the direction of the lanes compared to the transverse direction. hPSC-CFs cultured on micropatterned lanes and the 15 degree chevron pattern remodel the underlying ECM and produce fibers of collagen and fibronectin parallel to the feature direction. When co-cultured together on this pattern, ECM is produced by hPSC-CFs and hPSC-CMs display improved calcium kinetics and contractile strain. The ability to test factors individually and concomitantly in one biomimetic platform will lend new insights and aid in the directed maturation of immature hPSC-CMs, ultimately furthering our basic understanding of cardiac biology and providing novel platforms for drug discovery and toxicity testing.

The advent of human pluripotent stem cells (hPSCs) has provided many opportunities for studying and treating human diseases and testing human cells for the development of therapeutic drugs. Numerous studies have been conducted to develop differentiation protocols to derive specific cell types from hPSCs. Despite recent successes in the generation of various cell types with high yield, several challenges remain such as scaling differentiation to clinically and commercially relevant levels with minimal heterogeneity and creating a physiologically relevant cellular microenvironment in vitro for disease modeling and drug screening. However, these hurdles are not easily addressed with the conventional settings for hPSC culture and differentiation. In this project, we suggest proof-of-concept engineering strategies to genetically engineer hPSCs for reduced differentiation heterogeneity, spatially pattern differentiating hPSCs to control the cellular microenvironment, and engineer coatings to allow for adhesion and differentiation of hPSCs. We specifically focused on hPSC-derived cardiomyocytes (CMs) as this is a promising cell type that can be used to treat cardiac diseases responsible for most morbidity and mortality worldwide and to test cardiac toxicity of newly developed drugs. In chapter 1, we summarize the inductive factors that have been used to develop CM differentiation protocols for the last two decades. Then, in chapter 2, we present a cell-intrinsic control for CM differentiation to reduce variability caused by cell-extrinsic inductive factors during large-scale production. Cell-intrinsic control combines a genetic sensor that recognizes the cell's differentiation state and an actuator that induces cell specification into the next developmental stage. Using CRISPR-based genome editing, the T/Brachyury promoter was inserted in the genome of hPSCs as a genetic sensor that recognizes mesodermal state. Downstream of this promoter was the beta-catenin short-hairpin RNA and dCas9-KRAB targeting beta-catenin which served as genetic actuators to downregulate Wnt signaling pathway for specification of mesoderm into cardiac mesoderm. Even though we did not observe any significant enhancement in CM differentiation efficiency for either actuator, we found that the expression of dCas9-KRAB by the T/Brachyury promoter induced temporal downregulation of beta-catenin and Wnt signaling. As an alternative approach, MESP1, a cardiac lineage transcription factor, was used as an actuator, and we observed an increase in CM differentiation efficiency. Then, in chapter 3, we demonstrate control of the cellular microenvironment by spatially controlling stem cell differentiation. Localized adsorption of morphogens including BMP4, noggin, activin A, and Wnt3a were patterned to induce spatially controlled mesendoderm, endoderm, CM, and epicardial cell (EpiC) differentiation from hPSCs and hPSC-derived cardiac progenitors (CPCs). Importantly, patterned CM and EpiC co-differentiation allowed investigation of interactions between these cells in a spatially controlled manner and demonstrated improved alignment of CMs, an important metric of maturity and coordinated function, in proximity to differentiating EpiCs. Finally, in chapter 4, we improve hPSC cell adhesion by poly(norepinephrine) (pNE)-mediated surface modification to support extracellular matrix (ECM) protein coating on various materials. The pNE modification enhanced immobilization of ECM proteins and subsequent hPSC culture on polydimethylsiloxane (PDMS) and a non-adhesive hydrophilic surface. When the pNE coating was pre-patterned on the non-adhesive substrate, hPSCs and CPCs were spatially patterned on the coated region. We expect that this strategy can be used for the growth and differentiation of hPSCs on different substrates including tissue engineering scaffolds for creating three-dimensional or ECM-like structural microenvironments as well as for CM patterning to study effects of different spatial patterns of CM function. Collectively, these engineering strategies will contribute to expanding the potential applications of hPSC-derived CMs in regenerative medicine, drug screening, and disease modeling. We also expect the proposed strategies will provide new platform techniques and concepts not only for CM differentiation but also for the differentiation of hPSCs into other cell types.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.