A framework for developing sex-specific engineered heart models.

The convergence of tissue engineering and patient-specific stem cell biology has enabled the engineering of in vitro tissue models that allow the study of patient-tailored treatment modalities. However, sex-related disparities in health and disease, from systemic hormonal influences to cellular-level differences, are often overlooked in stem cell biology, tissue engineering and preclinical screening. The cardiovascular system, in particular, shows considerable sex-related differences, which need to be considered in cardiac tissue engineering. In this Review, we analyse sex-related properties of the heart muscle in the context of health and disease, and discuss a framework for including sex-based differences in human cardiac tissue engineering. We highlight how sex-based features can be implemented at the cellular and tissue levels, and how sex-specific cardiac models could advance the study of cardiovascular diseases. Finally, we define design criteria for sex-specific cardiac tissue engineering and provide an outlook to future research possibilities beyond the cardiovascular system.

Drawing Inspiration from Developmental Biology for Cardiac Tissue Engineers.

A sound understanding of developmental biology is part of the foundation of effective stem cell-derived tissue engineering. Here, the key concepts of cardiac development that are successfully applied in a bioinspired approach to growing engineered cardiac tissues, are reviewed. The native cardiac milieu is studied extensively from embryonic to adult phenotypes, as it provides a resource of factors, mechanisms, and protocols to consider when working toward establishing living tissues in vitro. It begins with the various cell types that constitute the cardiac tissue. It is discussed how myocytes interact with other cell types and their microenvironment and how they change over time from the embryonic to the adult states, with a view on how such changes affect the tissue function and may be used in engineered tissue models. Key embryonic signaling pathways that have been leveraged in the design of culture media and differentiation protocols are presented. The cellular microenvironment, from extracellular matrix chemical and physical properties, to the dynamic mechanical and electrical forces that are exerted on tissues is explored. It is shown that how such microenvironmental factors can inform the design of biomaterials, scaffolds, stimulation bioreactors, and maturation readouts, and suggest considerations for ongoing biomimetic advancement of engineered cardiac tissues and regeneration strategies for the future.

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems.

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

Robustness of the Dorsal morphogen gradient with respect to morphogen dosage.

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.

Spatiotemporal control of gene expression boundaries using a feedforward loop.

BACKGROUND: A feedforward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen-mediated tissue patterning. However, most morphogen gradients also evolve in time. RESULTS: We studied the spatiotemporal behavior of a coherent FFL in two contexts: (a) a generic, oscillating morphogen gradient and (b) the dorsal-ventral patterning of the early Drosophila embryo by a gradient of the NF-κB homolog dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node-phase difference and noise filtering-that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the dorsal gradient model, we also found that proper target gene expression was not possible without including the effect of maternal pioneer factor Zelda. CONCLUSIONS: An FFL buffers fluctuation to changes in the morphogen signal ensuring stable gene expression boundaries.