Safety and Efficacy of Dry Eye Intelligent Therapeutic Device in the Treatment of Meibomian Gland Dysfunction in Rabbits.

PURPOSE: To assess the safety and efficacy of the dry eye intelligent therapeutic device in rabbits with meibomian gland dysfunction. METHODS: The meibomian gland dysfunction-afflicted rabbits were subjected to treatment using the dry eye intelligent therapeutic device. Various parameters, including eyelid margin, meibomian gland opening, redness, meibomian gland area, keratoconjunctival fluorescence staining, and intraocular pressure, were examined and analyzed using an ocular surface comprehensive examination instrument, slit lamp, and tonometer at corresponding times points. Hematoxylin and eosin staining was performed to examine the mucosal epithelium and meibomian gland. RESULTS: In this study, eyelid margin congestion and meibomian gland opening obstruction were significantly improved after 3 weeks and 4 weeks of treatment, respectively (p < .01, p < .05). The treatment group showed a significant increase in tear meniscus height after 2 weeks, 3 weeks and 4 weeks of treatment (p < .001, p < .01, p < .05). No significant changes were noted in meibomian gland area, redness, intraocular pressure, and keratoconjunctival fluorescence staining of rabbits before and after treatment. Hematoxylin and eosin staining revealed a complete structure of mucosal epithelium and meibomian gland in the treatment group and that the expansion of the blocked meibomian gland duct was reduced. CONCLUSION: The utilization of the dry eye intelligent therapeutic device in treating meibomian gland dysfunction-afflicted rabbits exhibits potential promising safety, efficacy, and overall benefits, thereby offering a novel alternative for managing meibomian gland dysfunction patients in clinical settings.

The hiPSC-derived corneal endothelial progenitor-like cell recovers the rabbit model of corneal endothelial dystrophy.

INTRODUCTION: Corneal endothelial dysfunction results in cornea opacity, damaging sightedness, and affecting quality of life. A corneal transplant is the current effective intervention. Due to the scarcity of donated cornea, such an unmet medical need requires a novel therapeutic modality. OBJECTIVES: Customizing patients' corneal endothelial progenitor cells with proliferative activity and lineage restriction properties shall offer sufficient therapeutic cells for corneal endothelial dystrophy. METHODS: The customized induced human corneal endothelial progenitor-like cell (iHCEPLC) was obtained through cell fate conversions starting from PBMC (peripheral blood mononuclear cell), hiPSC (human induced pluripotent stem cell), and hNCC (human neural crest cell), while it finally reached the iHCEPLC state via a series of induction. Several molecular diagnoses were applied to depict its progenitor state, including RNAseq, FlowCytometer, immunostainings, and rtPCR. Significantly, it can be induced to gain differentiation maturity through contact inhibition. In addition, a BAK-mediated rabbit model of corneal endothelial dystrophy was established in the present study to test the therapeutic effectiveness of the iHCEPLC. RESULTS: After inducing cell fate conversion, the specific HCEC markers were detected by rtPCR and immunostaining in iHCEPLC. Further, RNAseq was applied to distinguish its progenitor-like cell fate from primary human corneal endothelial cells (HECE). FlowCytometry profiled the heterogeneity subpopulation, consistently displaying a subtle difference from primary HCEC. A terminal differentiation can be induced in iHCEPLC, addressing its progenitor-like fate. iHCEPLC can restore the BAK-based rabbit model of corneal endothelial dystrophy. Immunohistochemistry verified that such acuity restoration of the BAK-treated cornea is due to the introduced iHCEPLC, and such therapeutic effectiveness is observed in the long term. CONCLUSION: Here, we demonstrated that customized iHCEPLC has long-term therapeutic efficacy. As a progenitor cell, our iHCEPLC has a restricted cell lineage nature and can proliferate in vitro, supporting sufficient therapeutic candidate cells. Due to the immune-privileged nature of the cornea, our iHCEPLC proves the principle of therapeutical feasibility in both autogenic and allogeneic modalities.

The role of long noncoding RNAs in ocular angiogenesis and vascular oculopathy.

BACKGROUND: Long noncoding RNAs (lncRNAs) are RNA transcripts over 200 nucleotides in length that do not code for proteins. Initially considered a genomic mystery, an increasing number of lncRNAs have been shown to have vital roles in physiological and pathological conditions by regulating gene expression through diverse mechanisms depending on their subcellular localization. Dysregulated angiogenesis is responsible for various vascular oculopathies, including diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and corneal neovascularization. While anti-VEGF treatment is available, it is not curative, and long-term outcomes are suboptimal, and some patients are unresponsive. To better understand these diseases, researchers have investigated the role of lncRNAs in regulating angiogenesis and models of vascular oculopathies. This review summarizes recent research on lncRNAs in ocular angiogenesis, including the pro-angiogenic lncRNAs ANRIL, HOTAIR, HOTTIP, H19, IPW, MALAT1, MIAT, NEAT1, and TUG1, the anti-angiogenic lncRNAs MEG3 and PKNY, and the human/primate specific lncRNAs lncEGFL7OS, discussing their functions and mechanisms of action in vascular oculopathies.

Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation.

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Isolation and Identification of Limbal Niche Cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 °C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 °C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFRß). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFRß, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were 68%, 99%, 20%, and 3% in BMMSCs. The standard process for LNC isolation and identification could provide a reliable laboratory basis for the widespread use of LNCs.

3% diquafosol sodium eye drops in Chinese patients with dry eye: a phase IV study.

Introduction: The efficacy and safety of 3% diquafosol sodium eye drops in Chinese patients with dry eye in the real-world setting remains unclear. Methods: 3099 patients with dry eye symptoms were screened according to Asia Dry Eye Society latest recommendation. Among them, 3000 patients were enrolled for a phase IV study. We followed up with multiple clinical characteristics including corneal fluorescein staining, tear break up time, Schirmer's tests, visual acuity, intraocular pressure, and others. The follow ups were performed at baseline, 2 weeks and 4 weeks after treatment. Results: Based on the results of corneal fluorescein staining and tear break up time, all age and gender subgroups exhibited obvious alleviation of the symptoms among the patients with dry eye, and the data in elderly group showed the most significant alleviation. All the adverse drug reactions (ADRs, 6.17%) were recorded, among which 6% local ocular ADRs were included. Meanwhile, mild ADRs (91.8%) accounted for the most. Most of the ADRs (89.75%) got a quick and full recovery, with an average time at 15.6 days. 1.37% of patients dropped out of the study due to ADRs. Discussion: The use of 3% diquafosol sodium eye drop is effective and safe in the treatment of dry eye, with a low incidence of ADRs showing mild symptoms. This trial was registered at Chinese Clinical Trial Registry ID: ChiCTR1900021999 (Registration Date: 19/03/2019).

Diagnosis of fungal keratitis caused by Nectria haematococca through next-generation sequencing: review of literature and report of three cases.

OBJECTIVES: Fungal keratitis (FK) is a kind of serious corneal infection and penetrating keratoplasty (PKP) is needed when medical therapy fails. Although Nectria haematococca is found as endophytes in the roots of some plant species, there has been no report of N. haematococca infection in human. METHODS: We reviewed 46 patients who underwent PKP due to FK in our hospital from July 2021 to December 2021, and there were three patients who had relapsed. The next-generation sequencing revealed that all three corneas were infected with N. haematococca. RESULTS: Based on the ocular manifestation and treatment course of three cases, we summarize the characteristics of N. haematococca FK: the scope of corneal infection was widespread with severe hypopyon. The effect of local use of fluconazole and voriconazole was not ideal, and PKP was the main treatment. Even after a large-scale corneal lesion resection, the lesion may recur. The recurrence occurred primarily in the second week after PKP. CONCLUSION: This is the first clinical report of N. haematococca infection in humans. Compared with the other currently known FK caused by the Fusarium solani species complex, N. haematococca keratitis is more severe and more likely to recur.

Development and multi-center validation of machine learning model for early detection of fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is a leading cause of corneal blindness in developing countries due to poor clinical recognition and laboratory identification. Here, we aimed to identify the distinct clinical signature of FK and develop a diagnostic model to differentiate FK from other types of infectious keratitis. METHODS: We reviewed the electronic health records (EHRs) of all patients with suspected infectious keratitis in Beijing Tongren Hospital from January 2011 to December 2021. Twelve clinical signs of slit-lamp images were assessed by Lasso regression analysis and collinear variables were excluded. Three models based on binary logistic regression, random forest classification, and decision tree classification were trained for FK diagnosis and employed for internal validation. Independent external validation of the models was performed in a cohort of 420 patients from seven different ophthalmic centers to evaluate the accuracy, specificity, and sensitivity in real world. FINDINGS: Three diagnostic models of FK based on binary logistic regression, random forest classification, and decision tree classification were established and internal validation were achieved with the mean AUC of 0.916, 0.920, and 0.859, respectively. The models were well-calibrated by external validation using a prospective cohort including 210 FK and 210 non-FK patients from seven eye centers across China. The diagnostic model with the binary logistic regression algorithm classified the external validation dataset with a sensitivity of 0.907 (0.774, 1.000), specificity 0.899 (0.750, 1.000), accuracy 0.905 (0.805, 1.000), and AUC 0.903 (0.808, 0.998). INTERPRETATION: Our model enables rapid identification of FK, which will help ophthalmologists to establish a preliminary diagnosis and to improve the diagnostic accuracy in clinic. FUNDING: The Open Research Fund from the National Key Research and Development Program of China (2021YFC2301000) and the Open Research Fund from Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beijing Tongren Hospital, Beihang University &Capital Medical University (BHTR-KFJJ-202001) supported this study.

Progress of EGFL6 in angiogenesis and tumor development.

The epidermal growth factor (EGF) superfamily includes the protein 6 with an epidermal growth factor-like protein (EGFL6). EGFL6 has a signal peptide domain with an amino terminus and a MAM domain with a carboxy terminus. There are four whole EGF-like repeat regions and one partial EGF-like repeat region. Three of these regions include calcium-binding structures and an arg-gly-asp (RGD) integrin interaction motif. The epidermal growth factor-like (EGFL) and EGF domains have identical amino acid residues. Cell division, differentiation, mortality, cell adhesion, and migration are all affected by EGFL6. EGFL proteins are involved in a broad range of biological activities, making it important in tumor development and angiogenesis. We highlighted the latest development of EGFL6 research on tumor proliferation, invasion, and migration in this review.

Application Prospect and Preliminary Exploration of GelMA in Corneal Stroma Regeneration.

Corneal regeneration has become a prominent study area in recent decades. Because the corneal stroma contributes about 90% of the corneal thickness in the corneal structure, corneal stromal regeneration is critical for the treatment of cornea disease. Numerous materials, including deacetylated chitosan, hydrophilic gel, collagen, gelatin methacrylate (GelMA), serine protein, glycerol sebacate, and decellularized extracellular matrix, have been explored for keratocytes regeneration. GelMA is one of the most prominent materials, which is becoming more and more popular because of its outstanding three-dimensional scaffold structure, strong mechanics, good optical transmittance, and biocompatibility. This review discussed recent research on corneal stroma regeneration materials and related GelMA.

Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells.

Purpose: To investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro. Methods: MCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFRß. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control. Results: Homogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRß+. Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFRß transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRß was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63α than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 ± 57.91 µm), smaller in the LEPC group (125.71 ± 41.20 µm), and smallest in the MCEC group (109.39 ± 34.85 µm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups. Conclusions: The data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.

Risk Factors of Urrets-Zavalia Syndrome after Penetrating Keratoplasty.

Objective: The objective of this study was to analyze the clinical features and risk factors of Urrets-Zavalia syndrome (UZS) after penetrating keratoplasty (PKP). Methods: The medical records of 152 patients who underwent PKP at the Department of Ophthalmology, Tongji Hospital, between January 2014 and December 2016 were retrospectively reviewed. UZS was diagnosed based on pre- and post-operative pupillary findings. The relationships among the primary disease, postoperative intraocular pressure (IOP), and the incidence of UZS were statistically analyzed. The pupillary changes during the follow-up period were studied. Results: Among the 152 included patients, 23 were diagnosed with UZS, with an incidence of 15.13%. The primary diseases of the UZS patients were keratoconus (eight cases, 34.78%), viral keratitis (six cases, 26.08%), leukoma (four cases, 17.39%), fungal corneal ulcer (two cases, 8.70%), corneal endothelial decompensation (two cases, 8.70%), and corneal degeneration (one case, 4.35%). The incidence of UZS in keratoconus patients was higher than that in patients with fungal corneal ulcer (42.11% versus 6.25%, p = 0.003); In addition, the transient postoperative high IOP was not significantly related to the incidence of UZS in keratoconus patients in our study (p = 0.319). Twenty-one patients with UZS were followed up for >6 months, seven of whom (33.33%) recovered spontaneously (within the range of 48 days to 1.5 years). Conclusion: In our study, the incidence of UZS after PKP was 15.13%, and 33.33% of these patients recovered spontaneously. UZS may be more likely to occur in patients with keratoconus. Postoperative transient high IOP may increase the incidence of UZS after PKP for keratoconus.

Thickness Measurement of Endothelium-Descemet Membrane in Descemt Membrane Detachment Patients Using High-Definition Optical Coherence Tomography.

Purpose: (1) To measure the corneal endothelium-Descemet membrane (EDM) layer thickness in Descemet membrane detachment (DMD) patients in vivo using high-definition optical coherence tomography (HD-OCT), and to investigate its correlation with age. (2) To explore whether the detachment time will affect the EDM thickness. (3) To explore whether the EDM thickness of cornea with DMD was different from that without DMD. Participants: Patients with DMD were divided into three groups. Group 1 included twenty-three patients whose Descemet membrane (DM) was partial or complete detached from the corneal stroma after various ocular surgeries. Group 2 included eight patients from group 1 who underwent twice HD-OCT examination on different days before the DM reattached to the stroma. Group 3 included nine patients from group 1 who had clear grayscale boundary between the DM and stroma in HD-OCT images after DM reattachment. Methods: All patients underwent HD-OCT and EDM thickness was measured using Image -Pro Plus 6.0. In Group 1, regression analyses were used to evaluate the correlation between EDM thickness and age, and the thickness difference between the ≤50-year-old group and the >50-year-old group was analyzed by independent sample t-test. In Group 2, paired samples t-test was used to check whether detachment time would affect EDM thickness. In Group 3, paired samples t-test was used to check whether the EDM thickness of cornea with DMD was different from that without DMD. p < 0.05 was considered significant. Results: In Group 1, the EDM thickness measured on the first post-operative day was 27.8 ± 3.6 µm, and a positive correlation was found between EDM thickness and age (r = 0.619, p < 0.05). The EDM thickness of ≤50-year-old group and >50-year-old group were 23.9 ± 3.2 and 29.2 ± 2.6 µm, and there was a significant difference between the two groups (p = 0.001). In Group 2, the first measurement of EDM thickness was 27.5 ± 4.0 µm, the second measurement was 27.6 ± 4.2 µm, the interval between the two measurements was 2.1 ± 1.6 days, and there was no significant difference between the two measurements (p = 0.328). In Group 3, the EDM thickness with DM detachment was 28.3 ± 3.5 µm, with DM reattachment was 23.4 ± 2.4 µm, there was a significant difference between the two measurements (p = 0.002). Conclusions: The EDM thickness in the state of DMD is thicker than its actual thickness in normal cornea, and EDM thickness of the >50-year-old group is much thicker than that of the ≤50-year-old group.

Nicotinamide mononucleotide increases cell viability and restores tight junctions in high-glucose-treated human corneal epithelial cells via the SIRT1/Nrf2/HO-1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related corneal epithelial dysfunction is a severe ocular disorder; however, the effects of nicotinamide mononucleotide (NMN) on high-glucose (HG)-treated human corneal epithelial cells (HCECs) remain unclear. METHODS: We conducted an in-vitro study to examine the effects of NMN treatment on HG-treated HCECs. Cell viability was measured using trypan blue stain, mitochondrial membrane potential was measured using JC-1 stain, and intracellular reactive oxygen species and apoptosis assays were conducted using flow cytometry. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) immunofluorescence for tight junction examinations were conducted. Immunoblot analyses were conducted to analyze the expression of silent information regulator-1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) of the SIRT1/Nrf2/HO-1 pathway. RESULTS: NMN increased cell viability by reducing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs. By analyzing the expressions of SIRT1, Nrf2, HO-1, NMN demonstrated protective effects via the SIRT1/Nrf2/HO-1 pathway. CONCLUSIONS: NMN increases cell viability by reversing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs, and these effects may be mediated by the SIRT1/Nrf2/HO-1 pathway.

Limbal Bio-Engineered Tissue Employing 3D Nanofiber-Aerogel Scaffold to Facilitate LSCs Growth and Migration.

Constrained by the existing scaffold inability to mimic limbal niche, limbal bio-engineered tissue constructed in vitro is challenging to be widely used in clinical practice. Here, a 3D nanofiber-aerogel scaffold is fabricated by employing thermal cross-linking electrospinned film polycaprolactone (PCL) and gelatin (GEL) as the precursor. Benefiting from the cross-linked (160 °C, vacuum) structure, the homogenized and lyophilized 3D nanofiber-aerogel scaffold with preferable mechanical strength is capable of refraining the volume collapse in humid vitro. Intriguingly, compared with traditional electrospinning scaffolds, the authors' 3D nanofiber-aerogel scaffolds possess enhanced water absorption (1100-1300%), controllable aperture (50-100 µm), and excellent biocompatibility (optical density value, 0.953 ± 0.021). The well-matched aperture and nanostructure of the scaffolds with cells enable the construction of limbal bio-engineered tissue. It is foreseen that the proposed general method can be extended to various aerogels, providing new opportunities for the development of novel limbal bio-engineered tissue.

Somatic Reprogramming-Above and Beyond Pluripotency.

Pluripotent stem cells, having long been considered the fountain of youth, have caught the attention of many researchers from diverse backgrounds due to their capacity for unlimited self-renewal and potential to differentiate into all cell types. Over the past 15 years, the advanced development of induced pluripotent stem cells (iPSCs) has displayed an unparalleled potential for regenerative medicine, cell-based therapies, modeling human diseases in culture, and drug discovery. The transcription factor quartet (Oct4, Sox2, Klf4, and c-Myc) reprograms highly differentiated somatic cells back to a pluripotent state recapitulated embryonic stem cells (ESCs) in different aspects, including gene expression profile, epigenetic signature, and functional pluripotency. With the prior fruitful studies in SCNT and cell fusion experiments, iPSC finds its place and implicates that the differentiated somatic epigenome retains plasticity for re-gaining the pluripotency and further stretchability to reach a totipotency-like state. These achievements have revolutionized the concept and created a new avenue in biomedical sciences for clinical applications. With the advent of 15 years' progress-making after iPSC discovery, this review is focused on how the current concept is established by revisiting those essential landmark studies and summarizing its current biomedical applications status to facilitate the new era entry of regenerative therapy.

Soft substrate stiffness modifies corneal epithelial stem cell phenotype through hippo-YAP/notch pathway crosstalk.

Corneal disease remains to be one of the leading causes of blindness in the world and limbal stem cell (LSC) therapy is a promising therapy for LSC deficiency, which is associated with the diseased corneal epithelium repair. Soft substrate could effectively promote the stemness maintenance of LSC and thus modification of cell culture substrate would help in the potential LSC deficiency therapy. Both Hippo-Yes-associated protein (YAP) and Notch pathway have been reported to affect the LSC function, however, the detailed mechanisms remain unclear. Instead of some soft but biologically toxic substrates, we present a hypothesis on the application of soft substrate generated by HA/PTX3, an FDA approved nontoxic drug, on the LSC culture in this current study. Soft substrate could help in the stemness maintenance and thus promote the LSC deficiency treatment. In more detailed mechanism detection, we hypothesize that soft substrate would block the activation of Hippo-YAP pathway and thus decrease the activity of Notch pathway. This proposed hypothesis should be evaluated by both a series of in-vitro experiments based on soft and stiff substrates and in-vivo treatment with LSC cultured in different conditions. Advanced experiments on related cellular behaviors and detailed molecular mechanisms would provide us more knowledge on the molecular mechanism detection as well as cell transplantation therapy.

Correlations Between Single-Photon Emission Computed Tomography Parameters of Salivary Glands and Dry Eye Tests in Patients With Sjögren's Syndrome.

OBJECTIVE: To evaluate the correlations between Single-Photon Emission Computed Tomography (SPECT) parameters of salivary glands and dry eye parameters in patients with Sjögren's syndrome (SS). METHODS: A total of 28 patients with SS participated in this prospective study. Dry eye assessments include tear film break-up time (TBUT), corneal fluorescein staining scoring (CFS), Schirmer's I test (SIT) examination and SPECT of salivary gland. The following quantitative parameters were derived from SPECT imaging for salivary glands: Uptake index (UI), the time needed to achieve the minimum counts after Vit C stimulation (Ts), and excretion fraction (EF). The relation between the aforementioned parameters and TBUT, CFS and SIT were analyzed with SPSS 22.0 software. RESULTS: All the 28 eyes of the 28 subjects were examined. The mean SIT was 6.04 ± 4.64 mm/5 min (0-18 mm/5 min); the mean CFS was 3.07 ± 2.65 (0-10) and the mean BUT was 2.11 ± 1.97 s (0-9 s). The mean EF value was 0.52 ± 0.12 (0.26-0.75) in parotid glands and 0.45 ± 0.10 (0.30-0.67) in submandibular glands, respectively. The mean UI value was 9.33 ± 1.68 (6.03-13.20) in parotid glands and 9.92 ± 1.48 (7.08-12.60) in submandibular glands, respectively. The mean Ts (min) was 5.32 ± 3.01 (2.00-12.00) in parotid glands and 11.09 ± 7.40 (2.00- 29.00 min) in submandibular glands, respectively. It was found that EF positively correlates with SIT in patients with SS (r = 0.499 and 0.426 in parotid glands and submandibular glands, with P < 0.05), while no significant correlation was found between the UI, Ts and CFS, TBUT (P > 0.05). CONCLUSIONS: The EF was positively correlated with SIT in patients with SS, it could reflex the dysfunction of salivary glands in SS patients. So, EF may be a valuable parameter for the diagnosis of SS patients with lacrimal gland secretion dysfunction.

Opening the Soul Window Manually: Limbal Tissue Scaffolds with Electrospun Polycaprolactone/Gelatin Nanocomposites.

Restricted by the difficulty in fabricating scaffolds suitable for cell proliferation, the use of ex vivo expanded limbal stem cell (LSC) for LSC transplantation, an effective treatment method for patients with limb stem cell deficiency (LSCD), is hard to be widely used in clinical practice. To tackle these challenges, a novel electrospun polycaprolactone (PCL)/gelatin nanocomposite is proposed to make 3D scaffolds for limbal niche cells (LNC) proliferation in vitro, which is a milestone in the treatment of diseases such as LSCD. PCL and gelatin in different weight ratios are dissolved in a mixed solvent, and then electrospinning and cross-linking are performed to prepare a scaffold for cell proliferation. The characterizations of the nanocomposites indicate that the gelatin content has a significant effect on its micro-morphology, thermal properties, crystallinity, degradation temperature, hydrophilicity, and mechanical properties. P8G2-C (PCL: gelatin = 80: 20, cross-linked), with smooth fibers and homogeneous pores, has better hydrophilicity, mechanical properties, and flexibility, so it can support LNC as cell proliferation assays revealed. This detailed investigation presented here demonstrates the feasibility of using PCL/gelatin nanocomposites electrospun fiber membranes as a limbus tissue engineering scaffold, which undoubtedly provide a new perspective for the development of tissue engineering field.