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Understanding tissue formation advances our understanding of the causes of disease and the obtained knowledge can be potentially applied to develop personalized interventions. However, to explore the underlying mechanisms that govern tissue formation, there is a high and unmet need to develop new technologies to characterize different types of biomolecules from early-stage embryonic precursor cells and their descendent cells during development. This dissertation discusses new technological advancements to facilitate multi-omic (proteomic and metabolomic) analysis to explore cell-to-cell differences and uncover mechanisms underlying tissue formation. The work presented herein illustrates the development of in vivo microsampling and single-cell mass spectrometry (MS) to uncover cell heterogeneity among embryonic cells. Additionally, this dissertation work studies the biological role of metabolites in cell fate determination by exploring the mechanisms underlying metabolite-induced cell fate change. Moreover, this work introduces a novel technique called MagCar developed to track and isolate tissue-specific cells at later stages, which enables studying temporal molecular changes to gain new information about tissue formation.

Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesion sites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stress fibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stress fiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells. These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as to be crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates.

Development of new nerve guides is required for replacing autologous nerve grafts for the repair of long gap defects after nerve injury. A nerve guide comprised only of electrospun fibers able to bridge a critical (15 mm) nerve gap in a rat animal model is reported for the first time. The nerve conduits are made of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), a biocompatible copolymer composed of alternating amorphous, hydrophilic poly(ethylene oxide terephthalate), and crystalline, hydrophobic poly(butylene terephthalate) segments. These guides show suitable mechanical properties, high porosity, and fibers aligned in the longitudinal axis of the guide. In vitro studies show that both neurites and Schwann cells exhibit growth alignment with PA fibers. In vivo studies reveal that, after rat sciatic nerve transection and repair with PEOT/PBT guides, axons grow occupying a larger area compared to silicone tubes. Moreover, after repair of limiting (10 mm) and critical (15 mm) nerve gaps, PEOT/PBT guides significantly increase the percentage of regenerated nerves, the number of regenerated myelinated axons, and improve motor, sensory, and autonomic reinnervation in both gaps. This nerve conduit design combines the properties of PEOT/PBT with electrospun structure, demonstrating that nerve regeneration through long gaps can be achieved through the design of instructive biomaterial constructs.

Development of new nerve guides is required for replacing autologous nerve grafts for the repair of long gap defects after nerve injury. A nerve guide comprised only of electrospun fibers able to bridge a critical (15 mm) nerve gap in a rat animal model is reported for the first time. The nerve conduits are made of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), a biocompatible copolymer composed of alternating amorphous, hydrophilic poly(ethylene oxide terephthalate), and crystalline, hydrophobic poly(butylene terephthalate) segments. These guides show suitable mechanical properties, high porosity, and fibers aligned in the longitudinal axis of the guide. In vitro studies show that both neurites and Schwann cells exhibit growth alignment with PA fibers. In vivo studies reveal that, after rat sciatic nerve transection and repair with PEOT/PBT guides, axons grow occupying a larger area compared to silicone tubes. Moreover, after repair of limiting (10 mm) and critical (15 mm) nerve gaps, PEOT/PBT guides significantly increase the percentage of regenerated nerves, the number of regenerated myelinated axons, and improve motor, sensory, and autonomic reinnervation in both gaps. This nerve conduit design combines the properties of PEOT/PBT with electrospun structure, demonstrating that nerve regeneration through long gaps can be achieved through the design of instructive biomaterial constructs.