Human-induced pluripotent stem cells (iPSCs) offer transformative potential for biomedical research, with iPSC-derived organoids providing more physiologically relevant models than traditional 2D cell cultures. Among these, brain organoids (BO) are particularly valuable for drug screening, disease modeling, and investigations into molecular pathways. Accurate representation of brain morphology is critical, as more complex organoid structures better mimic the human brain. Deep learning (DL) and machine learning (ML) approaches have become integral to analyzing organoid morphology, yet tools for comprehensive, time-resolved assessments are scarce. Here, we introduce BrAIn, a DL-based application for analyzing the developmental progression of BOs. BrAIn tracks their evolution from embryoid bodies (EBs) and quantifies parameters including area, Feret diameter, perimeter, roundness, and circularity. It also classifies budding and abnormal morphologies of 3D organoids and detects monolayer neural rosette structures, key features of neuronal differentiation. Designed with accessibility in mind, BrAIn provides a no-code interface, enabling researchers of all technical backgrounds to conduct advanced morphological analyses with ease. Our study demonstrates the application of BrAIn to evaluate the effects of different growth conditions-static, orbital shaker, and microfluidic chip-based-on BO development. Orbital shaker cultures resulted in the largest organoids, while chip-based systems achieved more homogeneous growth. Both conditions produced organoids with greater morphological complexity compared to static culture. BrAIn emerges as a robust, user-friendly tool to quantify BO development and explore how versatile growth conditions influence their morphology and maturation.

AIMS:This study aims to investigate the efficacy of drug repurposing using a corneal organoid model developed from patient-derived iPSCs and to elucidate the pathophysiology of Aniridia-Associated Keratopathy (AAK). MATERIALS AND METHODS:A 90-day stepwise differentiation protocol was used to generate corneal organoids from iPSC cell lines developed from aniridia patients and healthy control. The corneal organoids produced were characterized using histology, immunofluorescence, qPCR, western blot, and transcriptomics. Two known agents, Duloxetine and Ataluren, were tested for corneal organoids for the restoring PAX6 protein expression. KEY FINDINGS:Histological analyses showed that the corneal organoids had a similar architecture to the native corneal tissue. Corneal epithelial, stromal, and endothelial cell biomarker staining showed positive expressions. AAK corneal organoids exhibited features that indicate the AAK disease phenotype, such as thickening of the epithelial cell layers and decrease in expressions of PAX6, ΔNP63, and keratocan genes. An increase in PAX6 protein was observed in organoids produced from AAK1 after duloxetine treatment and in organoids produced from AAK2 following ataluren treatment. AAK3 did not respond to either agent, indicating that there was no mutation-specific drug activity. Transcriptomic analyses showed clear corneal differentiation and absence of retina or lens profile in organoids. SIGNIFICANCE:This study presents patient-specific organoid models for AAK using iPSCs and offers insight into mutation effects and PAX6 restoration following drug repurposing. The findings form the basis of personalized treatments for congenital aniridia.

The retinohypothalamic tract (RHT) is the primary pathway for circadian photoentrainment. Rodent models exhibit a significant translational gap for human physiology due to their nocturnal nature. To overcome this, we developed a functional human RHT assembloid by fusing human pluripotent stem cell (hPSC) derived retinal and hypothalamus organoids. Characterization revealed mature retinal brush borders and the preservation of melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) integrated via excitatory glutamatergic synapses. Multielectrode array (MEA) analysis confirmed synchronized network activity across the interface. The development of the human RHT assembloid represents a significant leap forward in chronobiology. The gold standard for circadian models self-sustained gene expression oscillations was demonstrated using a PER2::Luciferase reporter, showing robust 20-30 hour rhythms. This validates the hypothalamic component as a functional "clock in a dish". This platform provides a readout to screen drugs or test light-pulse effects on circadian phase, directly modeling jet lag or phase-shifting. Overall, this model offers a high-fidelity system for investigating human-specific chronobiological mechanisms in vitro. ### Competing Interest Statement The authors have declared no competing interest. TUBİTAK, 124Z226

Aniridia is a rare panocular disease caused by gene mutation in the PAX6, which is essential for eye development. Aniridia is inherited in an autosomal dominant manner, but its phenotype can vary significantly among individuals with the same mutation. Animal models, such as drosophila, zebrafish, and rodents, have been used to study aniridia through Pax6 deletions. Recently, patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro, providing new insights into therapeutic strategies. In this study, corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations, allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived samples. Untargeted metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts, with amino acid metabolism pathways being consistently enriched in AAK organoids. This study emphasizes the profound impact of AAK mutations on metabolism, particularly in amino acid biosynthesis and energy metabolism pathways.

Lacrimal glands (LGs) serve as pivotal exocrine glands crucial for protecting the ocular surface. Dysfunction in LG cell composition or secretion is implicated in dry eye disease (DED). While autophagy plays a vital role in tissue homeostasis in many organs, how it affects LG development and secretory function is not known. Here, we have undertaken a genetic study by utilizing autophagy-deficient human embryonic stem cells (hESCs) and differentiating them into LG-like organoids. Autophagy-deficient LG-like organoids exhibited improper development and secretion, along with increased protein aggregation, proliferation, and cell death. These phenotypes were associated with an accumulation of PAX6, a transcription factor crucial for brain and eye development, which we identified as an autophagy substrate. Pharmacological interventions with nicotinamide mononucleotide (NMN) and melatonin were able to rescue the cellular dysfunction in autophagy-deficient LG-like organoids. Together, our study highlights the role of autophagy in LG along with potential therapeutic interventions for DED.

Organoids are self-assembled 3D cellular structures that resemble organs structurally and functionally, providing in vitro platforms for molecular and therapeutic studies. Generation of organoids from human cells often requires long and costly procedures with arguably low efficiency. Prediction and selection of cellular aggregates that result in healthy and functional organoids can be achieved by using artificial intelligence-based tools. Transforming images of 3D cellular constructs into digitally processable data sets for training deep learning models requires labeling of morphological boundaries, which often is performed manually. Here, we report an application named OrganoLabeler, which can create large image-based data sets in a consistent, reliable, fast, and user-friendly manner. OrganoLabeler can create segmented versions of images with combinations of contrast adjusting, K-means clustering, CLAHE, binary, and Otsu thresholding methods. We created embryoid body and brain organoid data sets, of which segmented images were manually created by human researchers and compared with OrganoLabeler. Validation is performed by training U-Net models, which are deep learning models specialized in image segmentation. U-Net models, which are trained with images segmented by OrganoLabeler, achieved similar or better segmentation accuracies than the ones trained with manually labeled reference images. OrganoLabeler can replace manual labeling, providing faster and more accurate results for organoid research free of charge.

Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.

Tissue engineering (TE) is a field of science that combines biological, engineering, and medical sciences and allows the development of disease models, drug development and gene therapy studies, and even cellular or tissue -based treatments developed by engineering methods. The eye is an organ that is easily accessible and amenable to engineering applications, paving the way for TE in ophthalmology. TE studies are being conducted on a wide range of topics, including the tear film, eyelids, cornea, optic nerve, glaucoma, and retinal diseases. With the rapid scientific advances in the field, it seems that TE is radically modifying the management of ocular disorders.

The increasing contamination of water with antibiotics presents significant environmental and health hazards, leading to a rise in antibiotic-resistant microorganisms. This work focused on the removal of one common pharmaceutical contaminant, Amoxicillin (AMX) from water. The approach involved enhancing a microfluidic chip by integrating a poly vinylidene fluoride (PVDF) membrane, aiming the highest and most precise adsorption capacity. The designed microfluidic chip included optical pH and dissolved oxygen sensors. The sensors enabled real-time monitoring of the solution to guarantee efficient removal of AMX. To increase the efficiency of a PVDF membrane for AMX elimination, MnFe2O4 nanoparticles with particle size around 25 nm were utilized as modifier adsorbent. The chemical, morphological, and elemental content of the modified porous membrane was determined using FTIR, XRD, FESEM, and EDS characterizations and confirmed the loading of MnFe2O4 nanoparticles on the PVDF membrane. The BET analysis revealed that the structural alteration obtained using the electrospray approach greatly increased the membrane's surface area after adding the nanoparticles approximately from 11.09 to 80.87 m2g-1. The hydrophilicity and thermal stability of the membranes were assessed, showing their applicability for efficient filtering operations. The optical sensors enabled real-time monitoring of pH in the range of 6-8 and dissolved oxygen in the range of 80-120 %, ensuring optimal conditions for amoxicillin adsorption in high flow rates of 100 mu l/min, approximately 99 %. The comprehensive design of this system significantly improved the efficiency of removing the AMX by follows the Langmuir's model, and demonstrated the adaptability and effectiveness of microfluidic technology in decreasing pharmaceutical pollution in the water sources. Moreover, it offers a simple and quick approach for users in the membrane industry to assess the manufactured membrane, making the technology accessible and feasible for broad use.

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4–7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

Organ‐on‐a‐chip (OoC) systems allow the generation of microphysiological tissue models that can recapitulate key biological processes in healthy and diseased states. OoC bone models provide valuable tools to study cross‐cellular interactions that take place in bone‐related processes. Although few bone‐on‐a‐chip models have been proposed, structural and biological hierarchy to establish a functional unit is often lacking. Herein, a functional OoC‐based 3D bone co‐culture model is reported. This model comprises a highly porous β‐tricalcium phosphate (TCP) based scaffold that is seeded with primary osteoblast and osteoclast precursors. This engineered construct is formed and cultured dynamically inside an OoC platform for up to 21 days and exhibits a dense extracellular matrix (ECM). Further, cultured constructs are ectopically implanted in C57BL/6 mice for 8 weeks, then histological and tartrate‐resistant acid phosphatase (TRAP) analyses are carried out. These results demonstrate that both bone deposition and resorption processes are present in the bioengineered model. This study also has implications for understanding complex cellular cross‐talks occurring in the bone remodeling process.

Aqueous tears secreted by the lacrimal gland have vital importance in maintaining and protecting the ocular surface health. Serious complications such as corneal ulceration, ocular surface keratinization and permanent vision loss can be seen in aqueous deficiency type dry eye disease that develops as a result of irreversible damage in the lacrimal gland. Current treatment options offer only short-term temporary palliation to reduce pain and inflammation on the ocular surface with no long-term improvement in lacrimal gland function. In recent years, the cellular and molecular properties of the lacrimal gland have been better understood, and studies carried out in the field of regenerative medicine show promise for the principal treatment of serious aqueous deficiency dry eye disease. In partial lacrimal gland damage, in situ regeneration can be achieved by using stem cells in the tissue. In total gland damage, healing can occur as a result of transplantation of organoids developed from induced pluripotent stem cells (iPSC) thanks to the tissue engineering method. Here, it is aimed to review the appropriate cellular resources for regeneration and development of functional artificial lacrimal gland by comparing studies using in situ stem cells and iPSC.

The corneal endothelium is composed of a single layer of specialized endothelial cells, protecting, and nourishing the inner surface of the cornea. Corneal endothelial cells do not proliferate after birth and their number decrease with age. Trauma, inflammation, or surgical intervention can cause cell loss. When damage is extensive and the density of corneal endothelial cells decreases to a critical level, it results in corneal edema and vision loss. Besides them, when corneal endothelium has irreversible damage, the only treatment way is corneal transplantation. But there are some drawbacks such as finding donors, immune reactions, and the number of patients waiting on the transplantation lists for years. Tissue engineering approaches can provide promising alternatives for the regeneration of corneal endothelium tissue. Peptides can be used to modify and functionalize the scaffolds, allowing for the production of bioactive and biomimetic surfaces. Peptide-modified scaffold surfaces might direct and enhance the behaviors of cells. In this study, the aim was to functionalize the polycaprolactone (PCL) membranes with tissue-specific peptides and to characterize the peptide-conjugated membranes by Fourier-Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and X-ray Photoelectron Spectroscopy (XPS) analysis. The synthesized peptides were successfully conjugated on the PCL biomembranes.

Tissue engineering applications hold the promise to restore, repair, and improve tissue function either through in vivo cellular therapy or by the use of in vitro developed tissue constructs. The biggest burden of the field limiting its clinical applications is the scarce number of cell sources. Stem cells, with their self-renewal and differentiation capacities, serve as an excellent cell source for tissue engineering applications. To search for stem cells with suitable differentiation capacities and inducing their differentiation to certain lineages is critical. Biomaterials with tailored properties offer microenvironments capable of controlling stem cell behavior by mimicking the stem cell niche. In this chapter, we explain the importance of stem cells in tissue engineering by covering their self-renewal and differentiation properties, we summarize the types of stem cells, their sources and offer some examples from the recent literature that employ stem cells to generate various tissues.

The first choice for reconstruction of clinical-size bone defects consists of autologous bone flaps, which often lack the required mechanical strength and cause significant donor-site morbidity. We have previously developed biological substitutes in a rabbit model by combining bone tissue engineering and flap pre-fabrication. However, spontaneous vascularization was insufficient to ensure progenitor survival in the core of the constructs. Here, we hypothesized that increased angiogenic stimulation within constructs by exogenous VEGF can significantly accelerate early vascularization and tissue in-growth. Bone marrow stromal cells from NZW rabbits (rBMSC) were transduced with a retroviral vector to express rabbit VEGF linked to a truncated version of rabbit CD4 as a cell-surface marker. Autologous cells were seeded in clinical-size 5.5 cm3 HA scaffolds wrapped in a panniculus carnosus flap to provide an ample vascular supply, and implanted ectopically. Constructs seeded with VEGF-expressing rBMSC showed significantly increased progenitor survivival, depth of tissue ingrowth and amount of mineralized tissue. Contrast-enhanced MRI after 1 week in vivo showed significantly improved tissue perfusion in the inner layer of the grafts compared to controls. Interestingly, grafts containing VEGF-expressing rBMSC displayed a hierarchically organized functional vascular tree, composed of dense capillary networks in the inner layers connected to large-caliber feeding vessels entering the constructs at the periphery. These data constitute proof of principle that providing sustained VEGF signaling, independently of cells experiencing hypoxia, is effective to drive rapid vascularization and increase early perfusion in clinical-size osteogenic grafts, leading to improved tissue formation deeper in the constructs.

There is a rapidly growing interest in generation of 3D organotypic microtissues with human physiologically relevant structure, function, and cell population in a wide range of applications including drug screening, in vitro physiological/pathological models, and regenerative medicine. Here, we provide a detailed procedure to generate structurally defined 3D organotypic microtissues from cells or cell spheroids using acoustic waves as a biocompatible and scaffold-free tissue engineering tool.