Cell spray printing combined with Lycium barbarum glycopeptide promotes repair of corneal epithelial injury.

The corneal epithelium, located as the outermost layer of the cornea, is inherently susceptible to injuries that may lead to corneal opacities and compromise visual acuity. Rapid restoration of corneal epithelial injury is crucial for maintaining the transparency and integrity of the cornea. Cell spray treatment emerges as an innovative and effective approach in the field of regenerative medicine. In our study, a cell spray printing platform was established, and the optimal printing parameters were determined to be a printing air pressure of 5 PSI (34.47 kPa) and a liquid flow rate of 30 ml/h. Under these conditions, the viability and phenotype of spray-printed corneal epithelial cells were preserved. Moreover, Lycium barbarum glycopeptide (LBGP), a glycoprotein purified from wolfberry, enhanced proliferation while simultaneously inhibiting apoptosis of the spray-printed corneal epithelial cells. We found that the combination of cell spray printing and LBGP facilitated the rapid construction of multilayered cell sheets on flat and curved collagen membranes in vitro. Furthermore, the combined cell spray printing and LBGP accelerated the recovery of the rat corneal epithelium in the mechanical injury model. Our findings offer a therapeutic avenue for addressing corneal epithelial injuries and regeneration.

Muse cell spheroids have therapeutic effect on corneal scarring wound in mice and tree shrews.

Stem cell therapy holds promises for treating corneal scarring. Here, we use multilineage-differentiating stress-enduring (Muse) cells to study their differentiation and therapeutic potential for treating corneal injury. Muse cells were isolated from lipoaspirate, which presented biphenotype properties of both pluripotent stem cells and some mesenchymal stem cells. Muse cells expanded by about 100-fold from the initial seeding cell number to Muse spheroids with the maintenance of the Muse cell phenotype and high cell viability at 33 days by static spheroid culture. We revealed that Muse spheroids were activated by the dynamic rotary cell culture system (RCCS), as characterized by increased stemness, improved activity, and enhanced adherence. Gene and protein expression of the pluripotent markers OCT3/4, SOX2, and NANOG and of the proliferation marker KI67 in Muse spheroids cultured under RCCS were higher than those in the static group. These activated Muse spheroids enabled ready differentiation into corneal stromal cells (CSCs) expressing characteristic marker genes and proteins. Furthermore, implantation of Muse cells-differentiated CSCs (Muse-CSCs) laden assembled with two orthogonally stacked stretched compressed collagen (cell-SCC) in mouse and tree shrew wounded corneas prevented the formation of corneal scarring, increased corneal re-epithelialization and nerve regrowth, and reduced the severity of corneal inflammation and neovascularization. cell-SCC retained the capacity to suppress corneal scarring after long-distance cryopreserved transport. Thus, Muse cell therapy is a promising avenue for developing therapeutics for treating corneal scarring.

Integrated bioinformatic changes and analysis of retina with time in diabetic rats.

Diabetic retinopathy (DR) is the most common chronic complication of diabetes. It can cause impaired vision and even blindness. However, the pathological mechanism of DR is still unknown. In the present study, we use bioinformatic analysis to reveal the pathological changes of early DR in a streptozotocin (STZ) induced diabetes rat model. The dataset GSE28831 was downloaded from the Gene Expression Omnibus (GEO) database. To clarify the pathological mechanism of early DR, genes which were up-regulated (UP group) or down-regulated (DOWN group) over time were identified. One hundred eighty six genes in the UP group and 85 genes in the DOWN group were defined. There were in total 28 Gene ontology (GO) terms with a P value lower than 0.05 in UP group, including astrocyte development, neutrophil chemotaxis, neutrophil aggregation, mesenchymal cell proliferation and so on. In the DOWN group, there were totally 14 GO terms with a P value lower than 0.05, including visual perception, lens development in camera-type eye, camera-type eye development, bicellular tight junction and so on. Signaling pathways were analyzed with all genes in the UP and DOWN groups, and leukocyte transendothelial migration and tight junction were selected. Protein-protein interaction (PPI) network was constructed and six hub genes Diras3, Actn1, Tssk6, Cnot6l, Tek and Fgf4 were selected with connection degree ≥5. S100a8, S100a9 and Tek may be potential targets for DR diagnosis and treatment. This study provides the basis for the diagnosis and treatment of DR in the future.

Pathological molecular mechanism of symptomatic late-onset Fuchs endothelial corneal dystrophy by bioinformatic analysis.

Fuchs endothelial corneal dystrophy (FECD) is a degenerative disease characterized by corneal endothelial decompensation. FECD causes corneal stromal and epithelial edema and progressively develops into bullous keratopathy, which can eventually lead to blindness. However, the exact pathogenesis is unknown. In this study, we performed an in-depth bioinformatic analysis of the dataset GSE74123 to determine the differentially expressed genes (DEGs) of symptomatic late-onset FECD compared with a normal control. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis were used to analyze the pathological molecular mechanism of FECD. We found that cell senescence, reactive oxygen species (ROS), the extracellular matrix (ECM), epithelial-mesenchymal transition (EMT) and immune response-related genes play an important role in the pathological development of symptomatic late-onset FECD. In addition, we revealed that down-regulated IL-6, enhanced NF-κB activity and a suite of orchestrated chemokine responses induce fibrocyte differentiation from monocyte to dendritic cell maturation. PI3K plays a key role in the molecular mechanism of symptomatic late-onset FECD. This study enhances our understanding of the molecular mechanism of FECD pathogenesis and will improve the diagnostics and therapy of FECD patients in the future.

Cell-laden and orthogonal-multilayer tissue-engineered corneal stroma induced by a mechanical collagen microenvironment and transplantation in a rabbit model.

The development of functional therapies for corneal repair and regeneration is a pressing issue. Corneal stroma provides the principal functions of the cornea. However, because of the highly organized nature of the stromal matrix, the attempts to reproduce corneal stroma might follow a scar model. Here, we have developed a protocol for the efficient generation of a cell-laden and orthogonal-multilayer tissue-engineered (TE) corneal stroma, which is induced by the mechanical effects of compressed collagen (CC) or stretched compressed collagen (SCC). Within SCC, with applied compression and force extension, collagen microfibres and corneal stromal cells (CSCs) are arranged orderly, while collagen fibres and CSCs in CC are randomly arranged. Dehydrated SCC has higher tensile strength than dehydrated CC. Hydrated SCC has similar transparency with hydrated native corneal stroma. Compared with those cultured on tissue culture plates (TCP), down-regulation of the genes and proteins of cytoskeleton, activation, proliferation, collagen and TRPV4, up-regulation of proteoglycans, gap junction proteins and TRPA1 are in CSCs of CC and SCC. Moreover, SCC and CC grafts displayed biocompatibility and integration with host corneal tissue after rabbit intra-corneal stromal transplantation by wk 6 under slit lamp microscopy, in vivo confocal microscopy and histological examination. The SCC model facilitates the construction of physiological feature TE corneal stroma, which serves as a foundation for physiological TE construction of other tissues. STATEMENT OF SIGNIFICANCE: The development of functional therapies for corneal repair and regeneration is a pressing issue. Corneal stroma provides the principal functions of the cornea. Here, we have developed a protocol for the efficient generation of a cell-laden and orthogonal-multilayer tissue-engineered (TE) corneal stroma, which is induced by the mechanical effects of compressed collagen (CC) or stretched compressed collagen (SCC). These models facilitate the construction of physiological feature TE corneal stroma, which serves as a foundation for physiological TE construction of other tissues and helps to reverse fibrosis pathologies in general.

Generation of an iPS cell line via a non-integrative method using urine-derived cells from a patient with USH2A-associated retinitis pigmentosa.

We have established an induced pluripotent stem (iPS) cell line using urine-derived cells from a 27-year-old male patient with retinitis pigmentosa associated with point mutations in the USH2A gene. Feeder-free culture conditions and the integration-free CytoTune™-iPS 2.0 Sendai Reprogramming Kit were used.

Cocktail of Chemical Compounds and Recombinant Proteins Robustly Promote the Stemness of Adipose-Derived Stem Cells.

Induced pluripotent stem cells (iPSCs) from somatic cells can be reprogrammed to provide an unlimited cell resource showing great potential in disease modeling and regenerative medicine. However, the traditional method for reprogramming cells into iPSCs using genome-integrating retro- or lenti-viruses remain an obstacle for its application in clinical settings. We tried the possibility to generate pre-iPSCs from human adipose-derived stem cells (ADSCs) by nongenetic reprogramming using recombinant cell-penetrating proteins OCT4/KLF4/SOX2 (PTD-OKS) and the cocktail of small molecules (VCFZ). Our experimental results demonstrated that PTD-OKS in combination with VCFZ (VCFZ+OKS) could significantly enhance the stemness of ADSCs and easily get pre-iPSCs after 25 days treatments. The pre-iPSCs showed similar morphology to iPSCs, which were positive for alkaline phosphatase staining. Furthermore, RT-polymerase chain reaction analysis showed that VCFZ+OKS could significantly upregulate the expression of OCT4, KLF4, SOX2, and NANOG gene after 25 days treatment. And immunofluorescence staining also showed that the protein makers of pluripotent stem cell were positively expressed in VCFZ+OKS treated group. Our data suggest that nongenetic-mediated reprogramming from ADSCs may be a promising stem cell sources for cell therapy in the near future.

In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers.

Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers in vitro.