Effect of Platelet-Riched Plasma on Corneal Epithelium Regeneration during Its Chronic Erosion (Experimental Study)

Relevance. In a number of pathological conditions accompanied by defects of the basal membrane, such as chemical or thermal burn, mechanical trauma, Schegren’s syndrome, herpetic keratitis processes of corneal epithelization are slowed down, and sometimes epithelization does not occur at all. Slow epithelization or its complete absence creates conditions for infection, thinning, and sometimes perforation of the cornea. That is why the problem of chronic corneal erosions is very relevant. The most perspective method of treatment is the use of autologous platelet-riched plasma (PRP). Presence of platelet growth factors, adhesive molecules and cytokines in PRP allows to use it for acceleration of regeneration of corneal defects, and the presence of live platelets allows to refer this procedure to autologous cell transplantations.Objective: To evaluate the clinical and morphological features of corneal epithelium regeneration in response to the application of PRP in conditions of experimental chronic corneal erosion (ECCE).Materials and methods. Researches were carried out on 12 rabbits of Chinchilla breed (24 eyes). At first stage the model of ECCE was reproduced for all animals: local ultraviolet (UV) irradiation with the exposure time of 45 min. on the preliminary de-epithelized corneal surface. At the second stage all animals under study were divided into three groups (4 rabbits in each group). I-st main group (MG) was instillated with PRP at a rate of 1 drop / 1 min within 10 minutes (total of 10 drops) once, in the II MG treatment was carried out according to the above method, every day for 5 days. In the control group (CG) PRP treatment was not applied.Results. 45-min. UV irradiation causes persistent ECCE up to 30 days of the experiment and reverts only after surface vessels are ingrowed in the affected area. Single use of PRP has insufficient therapeutic effect. Instillation within 5 days accelerates the cornea reparative regeneration in ECCE conditions. The absence of epithelial defect, newly formed vessels and corneal clouding was registered on the 9th day. Histologically, on the 30th day a normal multilayer non-squamous epithelium was found, while the stroma had an organized structure with no signs of inflammation.Conclusions. Therapeutic effect of PRP in ECCE conditions is based on reforming of the normal «adhesion complex» between epithelium, olfactory membrane and multifactor stimulation of reparative regeneration due to growth factors located in it, adhesive molecules and cytokines, as a result of which the typical corneal epithelium is restored.

Sox9-expressing cells promote regeneration after radiation-induced lung injury via the PI3K/AKT pathway

Background Radiation-induced lung injury (RILI) is considered one of the most common complications of thoracic radiation. Recent studies have focused on stem cell properties to obtain ideal therapeutic effects, and Sox9 has been reported to be involved in stem cell induction and differentiation. However, whether Sox9-expressing cells play a role in radiation repair and regeneration remains unknown. Methods We successfully obtained Sox9CreER, RosatdTomato and RosaDTA mice and identified Sox9-expressing cells through lineage tracing. Then, we evaluated the effects of the ablation of Sox9-expressing cells in vivo. Furthermore, we investigated the underlying mechanism of Sox9-expressing cells during lung regeneration via an online single-cell RNA-seq dataset. Results In our study, we demonstrated that Sox9-expressing cells promote the regeneration of lung tissues and that ablation of Sox9-expressing cells leads to severe phenotypes after radiation damage. In addition, analysis of an online scRNA-Seq dataset revealed that the PI3K/AKT pathway is enriched in Sox9-expressing cells during lung epithelium regeneration. Finally, the AKT inhibitor perifosine suppressed the regenerative effects of Sox9-expressing cells and the AKT pathway agonist promotes proliferation and differentiation. Conclusions Taken together, the findings of our study suggest that Sox9-expressing cells may serve as a therapeutic target in lung tissue after RILI.

The immune response is a critical regulator of zebrafish retinal pigment epithelium regeneration

Loss of the retinal pigment epithelium (RPE) because of dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms driving RPE regeneration. Here, utilizing transgenic and mutant zebrafish lines, pharmacological manipulations, transcriptomics, and imaging analyses, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. After genetic ablation, the RPE express immune-related genes, including leukocyte recruitment factors such as interleukin 34. We demonstrate that macrophage/microglia cells are responsive to RPE damage and that their function is required for the timely progression of the regenerative response. These data identify the molecular and cellular underpinnings of RPE regeneration and hold significant potential for translational approaches aimed toward promoting a pro-regenerative environment in mammalian RPE.

The common YAP activation mediates corneal epithelial regeneration and repair with different-sized wounds

Regeneration/repair after injury can be endowed by adult stem cells (ASCs) or lineage restricted and even terminally differentiated cells. In corneal epithelium, regeneration after a large wound depends on ASCs (limbal epithelial stem cells, LESCs), whereas repair after a small wound is LESCs-independent. Here, using rat corneal epithelial wounds with different sizes, we show that YAP activation promotes the activation and expansion of LESCs after a large wound, as well as the reprogramming of local epithelial cells (repairing epithelial cells) after a small wound, which contributes to LESCs-dependent and -independent wound healing, respectively. Mechanically, we highlight that the reciprocal regulation of YAP activity and the assembly of cell junction and cortical F-actin cytoskeleton accelerates corneal epithelial healing with different-sized wounds. Together, the common YAP activation and the underlying regulatory mechanism are harnessed by LESCs and lineage-restricted epithelial cells to cope with corneal epithelial wounds with different sizes.

Sox9-expressing cells promote regeneration after radiation-induced lung injury via PI3K/AKT pathway

Background Radiation induced lung injury (RILI) is considered as one of the most common complications of thoracic radiation. Recent studies have focused on stem cells properties to obtain ideal therapeutic effects and Sox9 has been reported to be involved in stem cell induction and differentiation. However, whether Sox9-expressing cells play a role in radiation repair and regeneration remain unknown. Methods We successfully obtain SOX9CreER, RosatdTomato and RosaDTA mice and identify Sox9-expressing cells through lineage tracing assay. Then we evaluated the effects of the ablation of Sox9-expressing cells in vivo. Furthermore, we investigated the underlying mechanism of Sox9 expressing cells during lung regeneration via an online single cell RNA-seq dataset. Results In our study, we demonstrated Sox9-expressing cells promote regenerative of lung tissues and ablation of Sox9-expressing cells leads to severe phenotypes after radiation damage. In addition, analysis of online scRNA-seq dataset revealed an enrichment of PI3K/AKT pathway in Sox9-expressing cells during lung epithelium regeneration. Finally, AKT inhibitor Perifosine could suppress the regenerative effects of Sox9-expressing cells. Conclusions Taken together, our study suggests that Sox9-expressing cells may serve as a therapeutic target in the setting of lung tissue after RILI.

Retinal Pigment Epithelium Regeneration by Induced Pluripotent Stem Cells; Therapeutic and Modelling Approaches on Retinal Degenerative Diseases

Retinal Degenerative Diseases [RDDs] are irreversible ocular damages categorized as retinopathies. RDDs affects about 0.05% of individuals worldwide. The degenerations of RPE cells are involved in inherited and age-related RDDs. After the invention of induced Pluripotent Stem Cell [iPSC] by Yamanaka, a promising window has been opened to regenerative medicine and disease modeling. Retinal pigment epithelium [RPE] degeneration related-RDDs are also affected by iPSCs. IPSC-derived RPE cells created a novel method for treating the RPE degeneration related-RDDs and retinal diseases modeling regarding finding a new therapeutic approach or drug development. There are various studies based on iPSC-derived RPE cells reporting the investigation of the role of a specific mutation, protein, signaling pathway, etc., responsible for a type of RDD. Furthermore, iPSC-based RPE therapy is expanded to include some clinical trials. Despite the incredible growth rate in iPSC-based studies in RPE-related diseases, there are some challenges, i.e., teratoma formation potential of iPSCs, the highly-cost procedure of iPSC-based regeneration of RPEs, lack of a universal protocol or cellular product applicable in all patients, etc. This article reviews the iPSC-based RPE generation and their therapeutic applications, studies on RPE-related molecular and cellular pathophysiologic features of RDD in the iPSC-based models, future perspectives, and the challenges ahead.

Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration

The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.

Novel In Vivo Mouse Cryoablation Model to Explore Unique Therapeutic Approaches for Premalignant Columnar Lesions

Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett’s esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N2O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett’s esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ.

Fabrication and Biocompatibility of Electroconductive Silk Fibroin/PEDOT: PSS Composites for Corneal Epithelial Regeneration

The aim of this study was to develop matrices that can support human corneal epithelial cells and innervation by incorporating a conducting polymer, poly(3,4-ethylenedioxythiophene) poly(styrene sulfonate) (PEDOT:PSS), into silk fibroin (SF). Polyvinyl alcohol (PVA) was used as a crosslinking agent to enhance the mechanical properties of the matrices. The impact of PEDOT:PSS on the materials’ physical properties and cellular responses was examined. The electrical impedance of matrices decreased with increasing concentration of PEDOT:PSS suggesting improved electroconductivity. However, light transmittance also decreased with increasing PEDOT:PSS. Young’s modulus was unaffected by PEDOT:PSS but was increased by PVA. The viability of corneal epithelial cell on the matrices was unaffected by the incorporation of PEDOT:PSS except at the highest concentration tested 0.3% (w/v), which led to a cytotoxic response. These findings suggest that SF/PEDOT:PSS with a PEDOT:PSS concentration of 0.1–0.2% would be a suitable biomaterial for epithelium regeneration.