Peter Christalla

Terminal Differentiation, Advanced Organotypic Maturation, and Modeling of Hypertrophic Growth in Engineered Heart Tissue

Rationale: Cardiac tissue engineering should provide “realistic” in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth. Objective: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth. Methods and Results: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7–12) was preceded by a tissue consolidation phase (culture days 0–7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring. Conclusions: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.

Parthenogenetic stem cells for tissue-engineered heart repair

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

The Cardiogenic Niche as a Fundamental Building Block of Engineered Myocardium

Cardiac muscle engineering is evolving rapidly, aiming at the provision of innovative models for drug development and therapeutic myocardium. The progress in this field will depend crucially on the proper exploitation of stem cell technologies. Understanding the processes governing stem cell differentiation towards a desired phenotype and subsequent maturation in an organotypic manner will be key to ultimately providing realistic tissue models or therapeutics. Cardiogenesis is controlled by milieu factors that collectively constitute a so-called cardiogenic niche. The components of the cardiogenic niche are not yet fully defined but include paracrine factors and instructive extracellular matrix. Both are provided by supportive stromal cells under strict spatial and temporal control. Detailed knowledge on the exact composition and functionality of the dynamic cardiogenic niche during development will likely be instrumental to further advance cardiac muscle engineering. This review will discuss the concept of myocardial tissue engineering from the stem cell/developmental biology perspective and put forward the hypothesis of the cardiogenic niche as a fundamental building block of tissue-engineered myocardium.

Preservation of left ventricular function and morphology in volume-loaded versus volume-unloaded heterotopic heart transplants

Total mechanical unloading of the heart in classical models of heterotopic heart transplantation leads to cardiac atrophy and functional deterioration. In contrast, partial unloading of failing human hearts with left ventricular (LV) assist devices (LVADs) can in some patients ameliorate heart failure symptoms. Here we tested in heterotopic rat heart transplant models whether partial volume-loading (VL; anastomoses: aorta of donor to aorta of recipient, pulmonary artery of donor to left atrium of donor, superior vena cava of donor to inferior vena cava of recipient; n = 27) is superior to the classical model of myocardial unloading (UL; anastomoses: aorta of donor to aorta of recipient, pulmonary artery of donor to inferior vena cava of recipient; n = 14) with respect to preservation of ventricular morphology and function. Echocardiography, magnetic resonance imaging, and LV-pressure-volume catheter revealed attenuated myocardial atrophy with ~30% higher LV weight and better systolic contractile function in VL compared with UL (fractional area shortening, 34% vs. 18%; maximal change in pressure over time, 2,986 ± 252 vs. 2,032 ± 193 mmHg/s). Interestingly, no differences in fibrosis (Picrosirus red staining) or glucose metabolism (2-[18F]-fluoro-2-deoxy-D-glucose-PET) between VL and UL were observed. We conclude that the rat model of partial VL attenuates atrophic remodelling and shows superior morphological as well as functional preservation, and thus should be considered more widely as a research model.

Cardiac engraftment of genetically-selected parthenogenetic stem cell-derived cardiomyocytes.

Parthenogenetic stem cells (PSCs) are a promising candidate donor for cell therapy applications. Similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PSCs exhibit self-renewing capacity and clonogenic proliferation in vitro. PSCs exhibit largely haploidentical genotype, and as such may constitute an attractive population for allogenic applications. In this study, PSCs isolated from transgenic mice carrying a cardiomyocyte-restricted reporter transgene to permit tracking of donor cells were genetically modified to carry a cardiomyocyte-restricted aminoglycoside phosphotransferase expression cassette (MHC-neor/pGK-hygror) to permit the generation of highly enriched cardiomyocyte cultures from spontaneously differentiating PSCs by simple selection with the neomycin analogue G148. Following engraftment into isogenic recipient hearts, the selected cardiomyocytes formed a functional syncytium with the host myocardium as evidenced by the presence of entrained intracellular calcium transients. These cells thus constitute a potential source of therapeutic donor cells.