Stem cells within the early embryo have the incredible capacity to self-organize and form higher order structures that provide the basis for large scale tissue morphogenesis. Understanding how embryonic signals are shaped, perceived, and transduced by cells early in embryonic development is critical for progress in the field of tissue engineering, so that we are able to recapitulate these cues in vitro and grow cells and tissue constructs outside of the body for high-throughput and animal-free drug/toxicological testing and cell/tissue replacement and therapy. However, efforts to study signaling and stem cell specification early in human development have largely been hindered by significant morphological differences in embryonic development of model organisms and ethical limitations regarding human embryo research. Thus, there is a need for tractable in vitro models which closely resemble native embryonic tissue, in both structure and behavior. Selecting an appropriate in vitro model that is tailored to hypothesis testing is critical for deriving conclusions about in vivo development. In this dissertation, I present a review on engineering co-emergence in organoid models, along with two studies which use novel 2D and 3D pluripotent stem cell models to study cell decision and symmetry breaking events during two different stages of early human embryonic development. In Chapter 1, I discuss the current state of organoid engineering, and define stringent criteria for organoid models. I discuss how primitive patterns and asymmetries integrate with endogenous cellular response systems to create autonomous and highly complex patterns that serve as a blueprint for higher order structure formation. In Chapter 2, I introduce cell structure as a key parameter which defines cell migration, organization, and specification. Specifically, I discuss how silencing of the cell-cell junction protein, CDH1, in a subpopulation of pluripotent stem cells within an embryoid body mimics zona pellucida hatching prior to gastrulation and prompts robust segregation and organization of CDH1- cells, along with their spontaneous differentiation to extraembryonic lineages. In Chapter 3, I focus on another cell-cell junction protein, ZO1, whose role is to both partition the plasma membrane into apical and basolateral functional domains, and assemble transmembrane tight junction proteins which prevent paracellular diffusion of macromolecules between the apical- and basolateral-facing lumens. Specifically, I demonstrate that cellular polarity, maintained by ZO1 in pluripotent stem cells, is a key regulator of morphogen signaling and pattern formation in a 2D peri-gastrulation model. In Chapter 4, I discuss the implications of these studies for signaling, specification, and migration of stem cells during embryonic development.

Interneurons, the most abundant neuronal class in the CNS, are critical to transducing synaptic information within and between neural networks for proper neurologic function. In the hindbrain and spinal cord, ventral interneurons are key components of pattern generators that control respiration and locomotion. Disruption of the interneurons by disease or injury can disrupt the pattern generators and lead to debilitation. Elucidating how these interneuron populations develop, as well as their roles in pattern generation, can lead to potential repair strategies for damaged circuits. While murine models have provided insight on interneuron development and circuitry, human populations may develop and function differently. Human pluripotent stem cell (hPSC)-derived neural populations provide a source to study human development and circuitry when alternative tissues are unavailable. This dissertation describes three studies that derive caudal interneuron populations from hPSCs. In the first study, V2a interneurons, a population involved in respiratory control and left-right coordination are differentiated from hPSCs. The V2a interneurons appropriately mature and survive to form long extensions following transplantation into an uninjured murine spinal cord. In the second study, multiple interneuron populations critical to respiratory control are specified from hPSCs. This study demonstrates the ability to describe multiple tissue-specific populations from one combination of signaling molecules. In the final study, a respiratory organoid is described that contains populations critical to respiratory control. This model provides a platform to probe how disruption of neural networks leads to respiratory distress. Together, this work is the first to describe multiple interneuron populations from hPSCs. These caudal interneurons can be used to study human development, test new drugs, and establish therapies to reconnect neural networks following injury or disease. Additionally, these studies provided a systematic approach to co-emerge multiple neural populations that could be broadly applied to describe other neural populations.

Embryogenesis is a critical period in the developmental life cycle of organisms, but the processes that enable multicellular coordination of germ layer formation and the subsequent dynamics of tissue structure assembly are still poorly described at the single cell level. Recent advances in microscopy and computer vision have enabled whole embryo tracking of cell migration, lineage commitment, and tissue formation in model organisms such as fly, zebrafish, frog, and chick, but it is unknown to what extent these organisms recapitulate the complex processes of early human development. Direct interrogation of the development of post-implantation human embryos carries technical and ethical limitations that render in vivo study of human embryogenesis largely observational, but recent advances in human induced pluripotent stem cells (hiPSCs) derived organoid models provide a powerful in vitro platform to elucidate developmental processes. The four studies described in this dissertation developed biological and computational pipelines to study the interdependencies between cell migration, lineage specification, and morphogenesis during early human development. First, several automated cell tracking algorithms were designed and deployed to study individual cell migration events in models of mosaic pattering and differentiation. Sparsely labeled cell populations were tracked during pluripotency, yielding measures of cell migration pre-differentiation, then cell behavior change kinetics were quantified in a mosaic Wnt differentiation model, demonstrating that automated cell tracking can non-destructively detect signatures of cell fate transition. Second, collective cell migration was interrogated using a novel ensemble of deep neural cell networks to generate dense spatio-temporal descriptions of hiPSC colony behaviors. Dense cell tracks revealed colony position dependent cell-cell interactions, including correlated migration, changes in cell density, and coordinated multicellular structure formation. Cell behaviors were found to be dependent on substrate, media, and colony size, and striking behavioral transitions were detected during differentiation to germ lineages using multiple protocols, including divergent cell migration patterns induced by Wnt and BMP signaling that nonetheless resulted in convergent germ layer organization. These results demonstrate how robust multicellular pattern formation is achieved through integration of cell intrinsic, local, and global signals. Third, collective migration and body axis emergence was studied in an organoid model of gastrulation and neural tube formation, where initially round organoids spontaneously assembled an organizer like structure and then underwent several days of elongation. Automated tracking of multi-day time lapse videos and segmentation of culture images spanning weeks of differentiation, facilitated quantification of organoid growth, elongation, and stratification, enabling optimization of culture conditions to improve differentiation robustness and repeatability across multiple stem cell lines. Elongation was demonstrated to depend on the emergence of a subpopulation of neuromesodermal progenitors (NMPs) which formed signaling centers reminiscent of the node structures that direct early primitive streak formation in vivo. Ablation of the early NMP transcription factor TBXT did not abrogate tail formation, but rather caused multiple spontaneous extensions to form, suggesting a key role for TBXT expressing cells in enabling the spontaneous symmetry breaking events underlaying the formation of a head-tail axis. Fourth and finally, a mechanistic simulation of the observed TBXT symmetry breaking and tail axis formation events was developed. Cell-cell interaction forces, cell migration parameters, and cell growth were derived from 2D measurements of re-plated organoids, shown to explain observed 3D organoid structure, growth, and elongation rates, and then applied to a 3D particle simulation of tail formation. Coalescence of structures matching the TBXT+ organizers were found to depend on both percentage of TBXT+ cells and the directionality of cell migration, demonstrating that a combination of chemotaxis and population heterogeneity is sufficient to produce organizer-like structures from initially randomly mixed populations. Overall, these studies demonstrate the critical activity of cell migration in coordinating multicellular organization during early embryogenesis, and provide a kinetic, mechanistic platform to interrogate how tissues form at multiple scales across the developmental landscape.

Native cardiac tissue is comprised of many different cell types that work cooperatively for proper tissue function. Combining self-assembled tissue engineering strategies that provide a fully-defined platform to study pairwise interactions between different cardiac cell types, with human pluripotent stem cell (hPSC) technologies, such as robust differentiation strategies and genome editing capabilities, has enabled our comprehensive studies of heterotypic interactions between cardiomyocytes and various non-myocyte sources in order to determine the specific contributions of non-myocytes to cardiac microtissue fate and function. We approached these studies in a systematic manner with narrowing focus.

We first broadly tested the most commonly-used stromal cells in cardiac tissue engineering studies and found that the different sources of stromal cells (primary human-derived vs. stem cell-derived; from different types of primary tissues) were distinct in terms of their surface marker expression, morphometry, and gene expression. These differences carried over into their ability to support engineered cardiac tissue formation and function, where only primary human cardiac fibroblasts and primary human dermal fibroblasts paired with hPSC-cardiomyocytes resulted in microtissues with the most robust tissue self-assembly and advanced calcium handling function.

Since the tissue-specific cardiac fibroblasts were able to positively support cardiac microtissue culture, we further characterized the specific contributions of different types of non-myocytes (endothelial cells, fetal human cardiac fibroblasts, adult human cardiac fibroblasts) in the context of heterotypic cardiac microtissue phenotype and function, at the single-cell and tissue-level, respectively. We found that 1 week after tissue formation, the cardiac microtissues containing the cardiac fibroblasts displayed more mature calcium handling properties relative to the tissues that contained endothelial cells and the tissues made from only cardiomyocytes, and that the cardiomyocytes paired with the cardiac fibroblasts were transcriptionally distinct from cardiomyocytes from the other tissues. However, after extended culture duration (1 month), the distinction between cardiac microtissues with cardiac fibroblasts versus without was lost, with the cardiomyocytes exhibiting similar transcriptomic profiles and the tissues displaying similar calcium transients. Furthermore, at both time points, there were no discernable differences between the different age cardiac fibroblasts, potentially because the source (isolated from primary tissue) was a bigger mismatch with stem cell-derived cardiomyocytes than the ontogenic difference.

Inspired by the pairing of different technologies to assess single-cell-level phenotype in the context of microtissue-level function, we sought to further characterize individual cell properties within intact 3D microtissues in order to better link the single cell building blocks to tissue-level properties. We used light sheet fluorescence microscopy to quantify 3D heterotypic multicellular organization as well as identify individual cardiomyocyte functional heterogeneity within heterotypic cardiac microtissues. Overall, this study demonstrated that advanced imaging techniques can be a powerful tool to dissect complex heterotypic interactions without removing the cells from their 3D environment.

Lastly, to dig deeper into the mechanisms governing the heterotypic interactions between cardiomyocyte and non-myocytes in our 3D engineered microtissues, we first had to generate cardiac tissues made from entirely stem cell-derived cellular constituents in order to take advantage of the robust genome engineering strategies developed for hPSCs. We were able to generate entirely-isogenic tissues when two differentiation protocols for the derivation of cardiac fibroblasts were published in 2019. We evaluated the different hPSC-cardiac fibroblast subtypes generated by these protocols in our heterotypic cardiac microtissue platform and found that they behaved similarly to one another and to microtissues made with primary human fetal cardiac fibroblasts in their ability to quickly self-assemble into tissues and their calcium handing function. We then knocked down one of the most-cited gap junctions that connects cardiomyocytes and cardiac fibroblasts in the heart, connexin 43, in the hPSC-derived cardiac fibroblasts using an inducible CRISPR interference method. Heterotypic cardiac microtissues generated with the knockdown fibroblasts displayed diminished calcium handling function, indicating a potential role for cardiac fibroblast support of cardiomyocyte function. Further mechanistic understanding of the interactions between cardiac cell populations can be determined in a similar manner, within the context of our fully-defined, tailored microtissue platform.

Taken together, this body of work provides a basis for the study of multicellular heterotypic interactions in 3D engineered models of myocardial tissue from human pluripotent stem cells. Future studies can build on this work by generating more complex microtissue constructs (i.e. incorporating more than two cell types, or modulating the cell types) as well as modeling cardiac diseases in tissue format. Combining genome editing, advanced imaging, and next-generation sequencing technologies enables customizable generation and comprehensive characterization of the multicellular interactions within engineered heterotypic tissue constructs.