Qianying Gao

Generation of Retinal Ganglion–like Cells from Reprogrammed Mouse Fibroblasts

PURPOSE Somatic cells can be reprogrammed into an embryonic stem cell-like pluripotent state by Oct-3/4, Sox2, c-Myc, and Klf4. Sox2 as an essential reprogramming factor also contributes to the development of the eye and the retina. This study was conducted to determine whether induced pluripotent stem (iPS) cells express retinal progenitor cell (RPC)-related genes and whether iPS cells can directly differentiate into retinal ganglion cells (RGCs). METHODS Mouse iPS cells were induced by the ectopically expressed four factors in tail-tip fibroblasts (TTFs). The expression of RPC-related genes in iPS cells was analyzed by RT-PCR and immunofluorescence. iPS cells were induced to differentiate into RGCs by the addition of Dkk1 + Noggin (DN) + DAPT and overexpression of Math5. iPS-derived retinal ganglion (RG)-like cells were injected into the retina, and the eyes were analyzed by immunohistochemistry. RESULTS iPS cells inherently express RPC-related genes such as Pax6, Rx, Otx2, Lhx2, and Nestin. Overexpression of Math5 and addition of DN can directly differentiate iPS into retinal ganglion-like cells. These iPS-derived RG-like cells display long synapses and gene expression patterns, including Math5, Brn3b, Islet-1, and Thy1.2. Furthermore, inhibiting Hes1 by DAPT increases the expression of RGC marker genes. In addition, iPS-derived RG-like cells were able to survive but were unable to be integrated into the normal retina after transplantation. CONCLUSIONS The four factor iPS cell inherently expressed RPC-related genes, and the iPS cell could be further turned into RG-like cells by the regulation of transcription factor expression. These findings demonstrate that iPS cells are valuable for regeneration research into retinal degeneration diseases.

Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming

Human induced pluripotent stem cells (iPSCs) provide a valuable model for regenerative medicine and human disease research. To date, however, the reprogramming efficiency of human adult cells is still low. Recent studies have revealed that cell cycle is a key parameter driving epigenetic reprogramming to pluripotency. As is well known, retroviruses such as the Moloney murine leukemia virus (MoMLV) require cell division to integrate into the host genome and replicate, whereas the target primary cells for reprogramming are a mixture of several cell types with different cell cycle rhythms. Whether cell cycle synchronization has potential effect on retrovirus induced reprogramming has not been detailed. In this study, utilizing transient serum starvation induced synchronization, we demonstrated that starvation generated a reversible cell cycle arrest and synchronously progressed through G2/M phase after release, substantially improving retroviral infection efficiency. Interestingly, synchronized human dermal fibroblasts (HDF) and adipose stem cells (ASC) exhibited more homogenous epithelial morphology than normal FBS control after infection, and the expression of epithelial markers such as E-cadherin and Epcam were strongly activated. Futhermore, synchronization treatment ultimately improved Nanog positive clones, achieved a 15–20 fold increase. These results suggested that cell cycle synchronization promotes the mesenchymal to epithelial transition (MET) and facilitates retrovirus mediated reprogramming. Our study, utilization of serum starvation rather than additional chemicals, provide a new insight into cell cycle regulation and induced reprogramming of human cells.

Mitochondria-Targeted Peptide MTP-131 Alleviates Mitochondrial Dysfunction and Oxidative Damage in Human Trabecular Meshwork Cells

PURPOSE To investigate the antioxidative ability of a novel mitochondria-targeted peptide MTP-131 in immortalized human trabecular meshwork (iHTM) and glaucomatous human trabecular meshwork (GTM(3)) cell lines. METHODS Cultured iHTM and GTM(3) cells were pretreated with MTP-131 for 1 hour, and sustained oxidative stress was induced by subjecting TM cells to 200 μM hydrogen peroxide (H(2)O(2)) for 24 hours. Untreated cells and cells incubated with H(2)O(2) alone were used as controls. Lactate dehydrogenase (LDH) assay was used to determine cell viability. Changes of mitochondrial membrane potential (ΔΨm) and generation of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry and confocal microscopy. Activation of caspase 3 was quantified by Western blotting, and apoptosis was measured by flow cytometry. Release of cytochrome c and changes in cytoskeleton were analyzed by confocal microscopy. Data were analyzed with commercial data analysis software and P < 0.05 was considered to be statistically significant. RESULTS In both iHTM and GTM(3) cells, decrease of ΔΨm and elevation of intracellular ROS were detected after sustained oxidative stress induced by H(2)O(2). When cells were pretreated with MTP-131, the H(2)O(2)-induced mitochondrial depolarization was prevented; intracellular ROS, LDH release, and apoptosis were significantly decreased; release of cytochrome c from mitochondria to cytoplasm and activation of caspase 3 were inhibited. In addition, cytoskeleton changes caused by H(2)O(2) were also alleviated by MTP-131. CONCLUSIONS Mitochondria-targeted peptide MTP-131 could prevent both iHTM and GTM(3) cells from sustained oxidative stress induced by H(2)O(2).

Evaluation of the Flexibility, Efficacy, and Safety of a Foldable Capsular Vitreous Body in the Treatment of Severe Retinal Detachment

PURPOSE To determine the flexibility, efficacy, and safety of a novel foldable capsular vitreous body (FCVB) in the treatment of severe retinal detachment in human eyes. METHODS The study involved 11 patients with 11 severe retinal detachments. A standard three-port pars plana vitrectomy was performed, and the FCVB was triple-folded and implanted into the vitreous cavity. Balanced salt solution was then injected into the capsule of the FCVB to support the retina. The treated eyes were examined by ophthalmoscopy, fundus photography, and tonometry during a 3-month implantation period. B-scan ultrasonography, optical coherence tomography (OCT), ultrasound biomicroscopy (UBM), and electroretinogram (ERG) were also performed. The FCVB was removed and examined in the laboratory at the end of the 3-month treatment time. RESULTS The FCVB was easily implanted into the vitreous cavity through a 3-mm incision and was easily removed through a 2-mm scleral incision. Retinal reattachment was found in 8 (73%) of 11 eyes at the end of the 3-month treatment time. The fundus, B-scan, and OCT showed that the FCVB was well distributed in the vitreous cavity and evenly supported the retina. IOP and visual acuity in the FCVB-treated eyes did not show a significant difference when compared with the preoperative measurements. UBM showed that the FCVB smoothly contacted but did not crush the ciliary body. Laboratory examinations showed no significant inflammatory cells in the balanced salt solution, no decrease in spectral transmittance, and no blocking of tiny apertures from the FCVB after a 3-month implantation period. CONCLUSIONS The FCVB was shown to be flexible, effective, and safe as a vitreous substitute over a 3-month implantation time. (ClinicalTrials.gov number, NCT00910702.).

Technical standards of a foldable capsular vitreous body in terms of mechanical, optical, and biocompatible properties.

We previously proposed a new strategy to fabricate a novel foldable capsular vitreous body (FCVB) as a vitreous substitute and found that the FCVB was a very good replacement for closely mimicking the morphology and restoring the physiologic function of the rabbit vitreous body. The aim of this article was to assess the mechanical, optical, and biocompatible properties of a FCVB made from liquid silicone rubber. The mechanical properties show that the shore hardness is 37.80 degrees, the tear strength is 47.14 N/mm, the tensile strength is more than 7.28 MPa, and the elongation ratio is more than 1200%; in addition, the FCVB has 300 nm mili apertures in the capsule. The optical properties reveal that transmittances are 92%, hazes are 5.74%, and spectral transmittance is 97%. The transmittance mission is 2.3% and can sustain a 1500 mW, 0.2 s, 532 nm green laser. The biocompatible properties are shown in the stable extracts experiment, no significant fever, good genetic safety, and no structural abnormality or apoptosis in the cornea, ciliary body, and retina over a 6-month observation period. These results indicate that the FCVB has good mechanical, optical, and biocompatible properties, and the assessment results can be recommended as the FCVB technical standards for industrial manufacturing and inspection.

Development and characterization of acellular porcine corneal matrix using sodium dodecylsulfate.

Purpose: The objective of this study was to produce a porcine corneal acellular matrix (ACM) and assess its possibility for biomedical applications. Methods: Porcine corneas were treated with various concentrations of sodium dodecylsulfate for different lengths of time. Optimal conditions for processing the ACM were noted regarding removal of all cellular components and retention of the spatial arrangement of the corneal stroma. The physical characteristics (including water absorption and light transmittance), biomechanics, and cytotoxicity of the ACM were also found to be conserved. Subsequently, ACM was transplanted into the interlaminar stroma of rabbit corneas. The transparency and structures of the collagen fibers were determined. Results: By immersing corneal tissues in isotonic buffer containing 0.1% sodium dodecylsulfate for 7 hours, we were able to produce an ACM whose cells were completely removed, without disrupting collagen layer structure. Although water absorption and light transmittance of the ACM decreased when compared with natural corneal stroma, ACM showed similar biomechanical properties and biocompatibility as natural ones. After xenotransplantation into rabbit corneal stromal layers for 4 weeks, both ACM and rabbit corneas showed complete transparency. Almost 1 year postoperatively, the corneas remained transparent with regular stromal structures and ACM appeared stable in situ without deliquescence or immunological rejection. Conclusions: A simple and valid method to produce decellularized corneal matrix has been successfully developed. These acellular matrices similar to natural corneas in structure, strength, and transparency have tremendous potential for corneal transplantation as ideal implants for donors and for tissue engineering applications as suitable scaffolds.

Sustained Mechanical Release of Dexamethasone Sodium Phosphate from a Foldable Capsular Vitreous Body

PURPOSE Since 300-nm-mili apertures were present in the capsule of the foldable capsular vitreous body (FCVB), the authors tested whether the FCVB could mechanically release dexamethasone sodium phosphate (DexP) from its capsule. METHODS In the in vitro study, DexP at concentrations of 0.25, 0.5, 1, 2, and 4 mg/mL in a balanced salt solution were injected into the capsules, which were immersed in cups of modified Franz diffusion cells. Two hundred microliters of liquid was aspirated at time intervals of 10, 20, 40, 60, 120, 180, 240, 300, and 360 minutes. In the in vivo study, the capsule was folded and implanted into the vitreous cavities of five rabbits. Approximately 0.6 mL DexP (2 mg/mL) was then injected into the capsule. An intravitreal injection with DexP was performed on another five rabbits as the control group. Aqueous humor was aspirated on days 1, 3, 7, 14, 28, and 42 after implantation. The DexP contents in the cups and aqueous humor were detected by HPLC-MS/MS. RESULTS FCVB released DexP in a time-dependent and dose-dependent manner in vitro with five dosages from 10 to 360 minutes. Especially in the 0.25 mg/mL DexP group, the content (y) had good linear relationships with time (x), as shown by y = 0.7635x + 10.205. The DexP contents in the aqueous humor were detected until day 28 and were undetectable on day 42. However, the DexP contents were detected only before day 3 in the controls. CONCLUSIONS FCVB can sustainably and mechanically release DexP by capsule apertures in a time-dependent and dose-dependent manner in addition to serving as a vitreous substitute.

A Novel Vitreous Substitute of Using a Foldable Capsular Vitreous Body Injected with Polyvinylalcohol Hydrogel

Hydrogels may be the ideal vitreous substitutes due to their wonderful physical features and biocompatibility. However, their drawbacks, short residence time, and biodegradation in vivo, have led to the fact that none of them have been approved for clinical use. In this study, we developed a novel approach of using a foldable capsular vitreous body (FCVB) injected with polyvinylalcohol (PVA) hydrogel as a vitreous substitute for long-term tamponade. The 3% PVA hydrogel that was cross-linked by gamma irradiation showed good rheological and physical properties and had no toxicity in vitro. After 180 days retention, the 3% PVA hydrogel inside FCVB remained transparent and showed good viscoelasticity without biodegradation and showed good biocompatibility and retina support. This new approach may develop into a valuable tool to improve the stability performance of PVA hydrogel as a vitreous substitute and to extend the application function of FCVB for long-term implantation in vitreous cavity.

A simple and evolutional approach proven to recanalise the nasolacrimal duct obstruction

Aim: To evaluate a new approach of recanalisation of nasolacrimal duct obstruction (RC-NLDO) in the treatment of the nasolacrimal duct obstruction (NLDO) and chronic dacryocystitis. Methods: 583 patients with 641 eyes suffering from NLDO and chronic dacryocystitis were enrolled in this study. The RC-NLDO was performed in 506 eyes, with 135 eyes undergoing external dacryocystorhinostomy (EX-DCR) as controls. Patient follow-up for 54 months was evaluated by symptoms, dye disappearance test, lacrimal irrigation and digital subtraction dacryocystogram. The RC-NLDO was also performed in 12 rhesus monkeys for histopathological examination. Results: The clinical success rates were 93.1% in 506 cases of RC-NLDO and 91.11% in 135 cases of EX-DCR. The success rates for second surgery were achieved in 85.19% on RC-NLDO and 40.0% on EX-DCR. No major intra- or postoperative complications were observed in the RC-NLDO group. The mean operative duration was 12.5 min for RC-NLDO and 40.3 min for EX-DCR (p<0.001). A pathological study in rhesus monkeys demonstrated that the RC-NLDO wounded epithelium in nasolacrimal duct healed completely within 1 month without granulation tissue formation. Conclusion: The findings demonstrate that the RC-NLDO is a simple and effective approach proven to recanalise the obstructed nasolacrimal duct with a comparable success rate to EX-DCR.

Carrier-free epithelial cell sheets prepared by enzymatic degradation of collagen gel

Limbal stem‐cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier‐free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier‐free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell–cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier‐free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration. Copyright