Tissue Engineering Strategies to Improve Post-MI Engraftment of hESC-Derived Cardiomyocytes

Thesis (Ph.D.)--University of Washington, 2016-12Transplantation of stem cell-derived cardiomyocytes is a promising strategy for repairing damaged cardiac muscle following a myocardial infarction. Our group and others have demonstrated both long-term engraftment and increased cardiac function after implantation in preclinical models using rodents and large animals. Despite this progress, there are still significant limitations to address in order to facilitate successful clinical translation of this therapy. Firstly, multiple delivery methods have been used to transplant stem cell-derived cardiomyocytes (hESC-cardiomyocytes) in rodents, including injecting cell suspensions and implanting engineered tissues. However, the ability of human cardiomyocytes to electrically and mechanically integrate with rodent myocardium using these delivery methods is not well understood. Secondly, current transplantation methods only retain a small fraction of implanted cells, leading to small graft size and an excess of cells needed for transplant. Here, we first conducted a comparative study to assess the engraftment and electromechanical integration of hESC-cardiomyocytes in the infarcted rat myocardium. This research demonstrated for the first time that human cardiomyocytes electrically integrate with the rat myocardium and beat in synchrony to rates over 6 Hz. We demonstrated that intramyocardially delivered cells (injected as a cell suspension or as cardiac micro-tissues) were electrically coupled to the host tissue, compared to no observed coupling when delivered as epicardial patches. All implant methods resulted in human myocardial grafts, however there was no improvement in graft area using these scaffold-free tissue engineering approaches compared to cell suspensions. To address this limitation, we designed an approach to improve engraftment and limit the number of cells required for implantation by promoting cardiomyocyte proliferation after transplantation. We developed a collagen-based hydrogel with the immobilized Notch ligand Delta-1, which was used in vitro to promote Notch signaling and increase cardiomyocyte proliferation by over 2-fold in engineered cardiac tissues. The optimized Notch-signaling hydrogel was then translated in vivo and used as a delivery vehicle for hESC-cardiomyocytes in the infarcted rat myocardium. This resulted in a 3-fold increase in cardiomyocyte proliferation and a 3-fold increase in graft size compared to controls. Taken collectively, the research in this dissertation highlights the potential of tissue engineering strategies to improve implantation of stem cell-derived cardiomyocytes, by promoting electromechanical integration and cell proliferation in preclinical models of myocardial infarction

Enhanced Electrical Integration of Engineered Human Myocardium via Intramyocardial versus Epicardial Delivery in Infarcted Rat Hearts.

Cardiac tissue engineering is a promising approach to provide large-scale tissues for transplantation to regenerate the heart after ischemic injury, however, integration with the host myocardium will be required to achieve electromechanical benefits. To test the ability of engineered heart tissues to electrically integrate with the host, 10 million human embryonic stem cell (hESC)-derived cardiomyocytes were used to form either scaffold-free tissue patches implanted on the epicardium or micro-tissue particles (~1000 cells/particle) delivered by intramyocardial injection into the left ventricular wall of the ischemia/reperfusion injured athymic rat heart. Results were compared to intramyocardial injection of 10 million dispersed hESC-cardiomyocytes. Graft size was not significantly different between treatment groups and correlated inversely with infarct size. After implantation on the epicardial surface, hESC-cardiac tissue patches were electromechanically active, but they beat slowly and were not electrically coupled to the host at 4 weeks based on ex vivo fluorescent imaging of their graft-autonomous GCaMP3 calcium reporter. Histologically, scar tissue physically separated the patch graft and host myocardium. In contrast, following intramyocardial injection of micro-tissue particles and suspended cardiomyocytes, 100% of the grafts detected by fluorescent GCaMP3 imaging were electrically coupled to the host heart at spontaneous rate and could follow host pacing up to a maximum of 300-390 beats per minute (5-6.5 Hz). Gap junctions between intramyocardial graft and host tissue were identified histologically. The extensive coupling and rapid response rate of the human myocardial grafts after intramyocardial delivery suggest electrophysiological adaptation of hESC-derived cardiomyocytes to the rat heart's pacemaking activity. These data support the use of the rat model for studying electromechanical integration of human cardiomyocytes, and they identify lack of electrical integration as a challenge to overcome in tissue engineered patches

Intramyocardial implants have connexin 43-positive junctions with host cardiomyocytes.

<p>Evidence of connexin 43-positive gap junction formation between host cardiomyocytes and hESC-cardiomyocyte grafts was found for cell grafts (A) and micro-tissue particle grafts (B). Boxed region in left column is shown at 4-fold magnification in the right column, highlighting the junctions between graft and host (white arrow heads). Patch implants showed connexin 43-positive regions within the patch (boxed region and right column) and no evidence of gap junction formation with the host, as hESC-cardiomyocytes were physically separated from the host myocardium by scar tissue (C). g, graft; s, scar, h, host. Scale bar = 50 μm.</p

Excitation response of stimulated hESC-cardiomyocytes in 2D culture <i>in vitro</i>.

<p>Spontaneous contraction rate was higher at 2 weeks with pacing at 1 and 6 Hz (*P < 0.05). Similarly, excitation threshold (ET) was significantly lower after 2 weeks of 1 and 6 Hz electrical pacing compared to unstimulated control. However, maximum capture rate (MCR) was not different among groups at any time point and all significant differences are lost after 4 weeks in culture.</p

Cardiomyocyte engraftment in injured rat hearts at 4 weeks.

<p>(A) Human cardiac grafts are identified by immunohistochemistry for GFP (brown, DAB) that binds to the GCaMP3 protein for the cell, micro-tissue particle (MTP), and patch groups. Hematoxalyin nuclear counterstain (blue). (B) Infarct scar area quantified by picrosirius red-positive area shows no difference between groups, normalized to left ventricular (LV) area at 4 weeks. (C) Graft size measured by GFP-positive graft area is normalized to LV area and is equivalent between groups (n = 8/group). (D) GFP-positive graft size declines with larger scar size for the cell grafts identified by histological analysis using Pearson correlation analysis (see text), but is weak and not significant in the micro-tissue particle or patch groups. (E) Micro-tissue particles (GFP, green) engrafted in the infarct region and lateral border within the septum of a rat heart are shown at 4 weeks stained for GFP (green) with DAPI (blue) nuclear stain. Topically applied dye (yellow-orange) marks the location of where an intramyocardial graft was detected by <i>ex vivo</i> imaging (arrow head). (F) Analysis of graft distribution in the heart indicates that cells and micro-tissue particles engraft in the scar, border zone, and healthy myocardium with equal distribution, while patch implants are found on the epicardium.</p

Formation of hESC-derived cardiac engineered tissues.

<p>(A) hESCs are differentiated into cardiomyocytes with high efficiency as characterized by flow cytometry analysis. An example cell population (85.5% single cell population, left) shows 81% expression of cardiac troponin T (cTnT, PE fluorescence, right) relative to isotype control (not shown). (B) Micro-tissue particles (MTPs) are formed by seeding approximately 1000 cells per microwell (left) and are easily removed from molds by a gentle media wash (right). (C) <i>In vitro</i> characterization of MTPs indicates highly defined particle diameter based on cell input number. (D) Cardiac engineered tissues have high cardiac purity as indicated by β-myosin heavy chain (β-MHC; brown, DAB) in MTPs (top) and cardiac patches (bottom). (E) Cardiac purity by β-MHC staining shows increasing purity with culture time. * P < 0.05 versus Day 1.</p

Summary of hESC-cardiomyocyte engraftment at 4 weeks by histology and <i>ex vivo</i> fluorescent imaging.

<p>No significant differences exist between treatment groups. MCR, maximum capture rate; NA, not applicable.</p

Human grafts contain striated cardiomyocytes.

<p>Confocal fluorescent imaging of engrafted hESC-cardiomyocytes at 4 weeks indicate that all three engraftment methods produce grafts that have high cardiac purity, as indicated by a double-positive stain for α-actinin (red) and GFP (green) with nuclear DAPI (blue), in representative images of all grafts. All grafts demonstrate sarcomere striations at higher magnification (far right). Scar tissue appears as the dark band separating the patch graft and host myocardium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131446#pone.0131446.g003" target="_blank">Fig 3</a> for picrosirius red labeled scar, black arrowhead) and this extends to the edges of the patch (outside the field of view; not shown). White arrowhead in patch image points to GFP⁺/α-actinin^- non-cardiac cells. g, graft; s, scar, h, host. Scale bar = 500 μm and 50 μm (far right column).</p

Assessment of human myocardial graft coupling to host heart by GCaMP3 <i>ex vivo</i> fluorescent imaging 4 weeks after implantation.

<p>(A) All micro-tissue particle and cell injection graft regions detected by <i>ex vivo</i> imaging are electrically coupled to the host, while no patch grafts were coupled to the host. Fluorescence signal of a GCaMP3 graft region vs. time is synchronized with the host electrocardiogram (ECG) for cell grafts at spontaneous rate (B; note that coupling persists during premature beats) and with 6 Hz stimulation (C) and for micro-tissue particle grafts at spontaneous rate (D) and 6 Hz stimulation (E). Epicardial patch grafts are easily detected during imaging but are uncoupled at a slower spontaneous rate (F).</p

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs

Summary: We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis. : Gunawardane and colleagues use CRISPR/Cas9 to deliver an excisable cassette to transcriptionally silent loci in hiPSCs, then accomplish excision of the cassette in a second step utilizing Cas9/CRISPR and the MMEJ and NHEJ DNA-repair pathways. Excision results in mEGFP tagging of the targeted loci. Upon differentiation, each of five tagged cell lines appropriately expresses a unique fluorescent fusion protein localized to the sarcomere in live cardiomyocytes. Keywords: CRISPR/Cas9, genome editing, cardiomyocyte differentiation, stem cells, iPSCs, MMEJ, live imaging, endogenous fluorescent tagging, mEGFP, HD