Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Force-dependent focal adhesion assembly and disassembly: A computational study.

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

Computational evidence for multi-layer crosstalk between the cadherin-11 and PDGFR pathways.

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell-cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR-α binding to extracellular cadherin-11 regions increases the PDGFR-α activity, whereas the interaction between PDGFR-ß and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between ß-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR-α and -ß. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.

Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

Complementary Supramolecular Functionalization Enhances Antifouling Surfaces: A Ureidopyrimidinone-Functionalized Phosphorylcholine Polymer.

Fibrosis of implants remains a significant challenge in the use of biomedical devices and tissue engineering materials. Antifouling coatings, including synthetic zwitterionic coatings, have been developed to prevent fouling and cell adhesion to several implantable biomaterials. While many of these coatings need covalent attachment, a conceptually simpler approach is to use a spontaneous self-assembly event to anchor the coating to a surface. This could simplify material processing through highly specific molecular recognition. Herein, we investigate the ability to utilize directional supramolecular interactions to anchor an antifouling coating to a polymer surface containing a complementary supramolecular unit. A library of controlled copolymerization of ureidopyrimidinone methacrylate (UPyMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) was prepared and their UPy composition was assessed. The MPC-UPy copolymers were characterized by 1H NMR, Fourier transform infrared (FTIR), and gel permeation chromatography (GPC) and found to exhibit similar mol % of UPy as compared to feed ratios and low dispersities. The copolymers were then coated on an UPy elastomer and the surfaces were assessed for hydrophilicity, protein absorption, and cell adhesion. By challenging the coatings, we found that the antifouling properties of the MPC-UPy copolymers with more UPy mol % lasted longer than the MPC homopolymer or low UPy mol % copolymers. As a result, the bioantifouling nature could be tuned to exhibit spatio-temporal control, namely, the longevity of a coating increased with UPy composition. In addition, these coatings showed nontoxicity and biocompatibility, indicating their potential use in biomaterials as antifouling coatings. Surface modification employing supramolecular interactions provided an approach that merges the simplicity and scalability of nonspecific coating methodology with the specific anchoring capacity found when using conventional covalent grafting with longevity that could be engineered by the supramolecular composition itself.

A single-cell RNA-seq analysis unravels the heterogeneity of primary cultured human corneal endothelial cells.

The cornea is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparency. CECs remain arrested in a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo-temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

Future directions in managing aniridia-associated keratopathy.

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies.

Elucidating the Corneal Endothelial Cell Proliferation Capacity through an Interspecies Transcriptome Comparison.

The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals, CECs are arrested in a non-proliferative state. Damage to these cells can compromise their function causing corneal opacity. Corneal transplantation is the current treatment for the recovery of clear eyesight, but the donor tissue demand is higher than the availability and there is a need to develop novel treatments. Interestingly, rabbit CECs retain a high proliferative profile and can repopulate the endothelium. There is a lack of fundamental knowledge to explain these differences. Gaining information on their transcriptomic variances could allow the identification of CEC proliferation drivers. In this study, human, sheep, and rabbit CECs are analyzed at the transcriptomic level. To understand the differences across each species, a pipeline for the analysis of pathways with different activities is generated. The results reveal that 52 pathways have different activity when comparing species with non-proliferative CECs (human and sheep) to species with proliferative CECs (rabbit). The results show that Notch and TGF-ß pathways have increased activity in species with non-proliferative CECs, which might be associated with their low proliferation. Overall, this study illustrates transcriptomic pathway-level differences that can provide leads to develop novel therapies to regenerate the corneal endothelium.

Optimization of Media Change Intervals through Hydrogels Using Mathematical Models.

Three-dimensional cell culture in engineered hydrogels is increasingly used in tissue engineering and regenerative medicine. The transfer of nutrients, gases, and waste materials through these hydrogels is of utmost importance for cell viability and response, yet the translation of diffusion coefficients into practical guidelines is not well established. Here, we combined mathematical modeling, fluorescent recovery after photobleaching, and hydrogel diffusion experiments on cell culture inserts to provide a multiscale practical approach for diffusion. We observed a dampening effect of the hydrogel that slowed the response to concentration changes and the creation of a diffusion gradient in the hydrogel by media refreshment. Our designed model combined with measurements provides a practical point of reference for diffusion coefficients in real-world culture conditions, enabling more informed choices on hydrogel culture conditions. This model can be improved in the future to simulate more complicated intrinsic hydrogel properties and study the effects of secondary interactions on the diffusion of analytes through the hydrogel.

Enhanced Microvasculature Formation and Patterning in iPSC-Derived Kidney Organoids Cultured in Physiological Hypoxia.

Stem cell-derived kidney organoids have been shown to self-organize from induced pluripotent stem cells into most important renal structures. However, the structures remain immature in culture and contain endothelial networks with low connectivity and limited organoid invasion. Furthermore, the nephrons lose their phenotype after approximately 25 days. To become applicable for future transplantation, further maturation in vitro is essential. Since kidneys in vivo develop in hypoxia, we studied the modulation of oxygen availability in culture. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, growth factor expression such as VEGF-A and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional vessel quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of VEGFA-189 and VEGFA-121, and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

Modular mixing of benzene-1,3,5-tricarboxamide supramolecular hydrogelators allows tunable biomimetic hydrogels for control of cell aggregation in 3D.

Few synthetic hydrogels can mimic both the viscoelasticity and supramolecular fibrous structure found in the naturally occurring extracellular matrix (ECM). Furthermore, the ability to control the viscoelasticity of fibrous supramolecular hydrogel networks to influence cell culture remains a challenge. Here, we show that modular mixing of supramolecular architectures with slow and fast exchange dynamics can provide a suitable environment for multiple cell types and influence cellular aggregation. We employed modular mixing of two synthetic benzene-1,3,5-tricarboxamide (BTA) architectures: a small molecule water-soluble BTA with slow exchange dynamics and a telechelic polymeric BTA-PEG-BTA with fast exchange dynamics. Copolymerisation of these two supramolecular architectures was observed, and all tested formulations formed stable hydrogels in water and cell culture media. We found that rational tuning of mechanical and viscoelastic properties is possible by mixing BTA with BTA-PEG-BTA. These hydrogels showed high viability for both chondrocyte (ATDC5) and human dermal fibroblast (HDF) encapsulation (>80%) and supported neuronal outgrowth (PC12 and dorsal root ganglion, DRG). Furthermore, ATDC5s and human mesenchymal stem cells (hMSCs) were able to form spheroids within these viscoelastic hydrogels, with control over cell aggregation modulated by the dynamic properties of the material. Overall, this study shows that modular mixing of supramolecular architectures enables tunable fibrous hydrogels, creating a biomimetic environment for cell encapsulation. These materials are suitable for the formation and culture of spheroids in 3D, critical for upscaling tissue engineering approaches towards cell densities relevant for physiological tissues.

Soft, Dynamic Hydrogel Confinement Improves Kidney Organoid Lumen Morphology and Reduces Epithelial-Mesenchymal Transition in Culture.

Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial-mesenchymal transition (EMT), whereas encapsulation in soft, stress-relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure-property-function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease-modeling applications.

Cadherin-11 Influences Differentiation in Human Mesenchymal Stem Cells by Regulating the Extracellular Matrix Via the TGFß1 Pathway.

For regenerative medicine, directing stem cell fate is one of the key aims. Human mesenchymal stem cells (hMSCs) are versatile adult stem cells that have been proposed for several clinical applications, making directing their fate of utmost importance. For most clinical applications, their differentiation toward the adipogenic lineage is an undesired outcome. Understanding the mechanisms that regulate hMSC commitment toward the adipogenic lineage might help open up new avenues for fine-tuning implanted hMSCs for regenerative medicine applications. We know that cadherin-11 is required for hMSC commitment to the adipogenic lineage; therefore, we sought to investigate the mechanisms through which cadherin-11 regulates adipogenic differentiation. We observed that hMSCs lacking cadherin-11 had decreased expression of type VI collagen and increased expression of fibronectin. We provide evidence of increased transforming growth factor beta 1 and the subsequent translocation of phosphorylated SMAD2/3 into the nucleus by cells that lack cadherin-11, which could be attributed to the changes in extracellular matrix composition. Taken together, our study implicates cadherin-11 in regulating extracellular matrix production and thereby helping improve cell- and material-based regenerative medicine approaches.

Cadherin-11 Regulates Cell Proliferation via the PDGFRß-ERK1/2 Signaling Pathway in Human Mesenchymal Stem Cells.

Controlling stem cell fate is the cornerstone of regenerative medicine. Cadherins have an important role in cell fate commitment and the function of cadherin-11 in the regulation of differentiation in human mesenchymal stem cells (hMSCs) has recently come to light. To better understand how cadherin-11 regulates hMSC behavior, we explored its interaction with receptor tyrosine kinases (RTK), an important family of proteins involved in a myriad of cellular functions. In this study, we provide evidence that cadherin-11, a cell adhesion protein expressed in hMSCs, regulates the activity of several RTKs, including PDGFRß and PDGFRα. By knocking down cadherin-11 we found that the changes in the RTK activity caused hyperactivation of the MAPK pathways, which were sustained through the phosphorylation and nuclear translocation of ERK1/2 and subsequently caused a decrease in cell proliferation. Together these results provide compelling evidence for the important role of the interaction of cadherin-11 and RTKs in the behavior of hMSCs.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

A comparative study of mesenchymal stem cells cultured as cell-only aggregates and in encapsulated hydrogels.

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs.

Approaches for corneal endothelium regenerative medicine.

The state of the art therapy for treating corneal endothelial disease is transplantation. Advances in the reproducibility and accessibility of surgical techniques are increasing the number of corneal transplants, thereby causing a global deficit of donor corneas and leaving 12.7 million patients with addressable visual impairment. Approaches to regenerate the corneal endothelium offer a solution to the current tissue scarcity and a treatment to those in need. Methods for generating corneal endothelial cells into numbers that could address the current tissue shortage and the possible strategies used to deliver them have now become a therapeutic reality with clinical trials taking place in Japan, Singapore and Mexico. Nevertheless, there is still a long way before such therapies are approved by regulatory bodies and become clinical practice. Moreover, acellular corneal endothelial graft equivalents and certain drugs could provide a treatment option for specific disease conditions without the need of donor tissue or cells. Finally, with the emergence of gene modulation therapies to treat corneal endothelial disease, it would be possible to treat presymptomatic patients or those presenting early symptoms, drastically reducing the need for donor tissue. It is necessary to understand the most recent developments in this rapidly evolving field to know which conditions could be treated with which approach. This article provides an overview of the current and developing regenerative medicine therapies to treat corneal endothelial disease and provides the necessary guidance and understanding towards the treatment of corneal endothelial disease.

Single cell transcriptomics reveals the heterogeneity of the human cornea to identify novel markers of the limbus and stroma.

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.