Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation.

Severe corneal injury is one of the main causes of loss of visual function. Mesenchymal stem cells (MSCs) have the ability to repair damaged cells in vivo. The present study aimed to explore whether MSCs could function as a cell therapy tool to replace traditional methods to treat corneal injury. CD44 + /CD105 + mesenchymal stem cells isolated from mouse amniotic fluid (mAF-MSCs) were injected into mice after cryoinjury to induce corneal endothelial cell injury. Histopathological assays indicated that mAF-MSCs could promote the growth of corneal epithelial cells, reduce keratitis, and repair the corneal damage caused by low temperature. cDNA microarray analysis revealed that the mAF-MSCs affected the expression patterns of mRNAs related to cell proliferation and differentiation pathways in the mice after transplantation. The results of quantitative real-time PCR and western blotting revealed that NAT12, NAT10, and the ETV4/JUN/CCND2 signaling axis were elevated significantly in the mAF-MSC-transplantation group, compared with those in the phosphate-buffered saline-treated groups. High performance liquid chromatography-mass spectroscopy results revealed that mAF-MSCs could promote mRNA N4-acetylcytidine (ac4C) modification and high expression of N-acetyltransferase in the eyeballs. RNA immunoprecipitation-PCR results showed that a specific product comprising Vegfa, Klf4, Ccnd2, Jun, and Etv4 mRNA specific coding region sites could be amplified using PCR from complexes formed in mAF-MSC-transplanted samples cross-linked with anti-ac4C antibodies. Thus, mouse amniotic fluid MSCs could repair the mouse corneal cold injury by promoting the ETV4/JUN/CCND2 signal axis activation and improving its stability by stimulating N4-acetylcytidine modification of their mRNAs.

Calcium Oscillatory Behavior and Its Possible Role during Wound Healing in Bovine Corneal Endothelial Cells in Culture.

In epithelial layers in culture, immediately after an injury a fast calcium wave (FCW) propagates from the wound borders toward the rest of the monolayer. We show here that similarly to other tissues, during the FCW in bovine corneal endothelial (BCE) cells in culture many cells exhibit calcium oscillations mediated by IP3 signaling. In this study we perform a detailed characterization of this oscillatory behavior and explore its possible role in the process of wound healing. In previous work we showed that, in BCE cells in culture, the healing cells undergo two stages of caspase-dependent apoptosis, at approximately two and eight hours after wounding. We determined that inhibition of the FCW greatly increases the apoptotic rate of the two stages, suggesting that the wave prevents excessive apoptosis of the healing cells. Taking this into account, we investigated the possible participation of the calcium oscillations during the FCW in apoptosis of the healing cells. For this, we employed ARL-67156 (ARL), a weak competitive inhibitor of ecto-ATPases, and the calcium chelator EGTA. We show here that, in healing BCE cells, ARL enhances cellular calcium oscillations during the FCW, while EGTA decreases oscillations. We found that ARL produces a significant decrease (to about half the control value) in the apoptotic index of the first stage of apoptosis, while EGTA increases it. Neither drug noticeably affects the second stage. We have interpreted the effect of ARL on apoptosis as due to the maintenance of moderately risen ATP levels during the FCW, which is in turn the cause for the enhancement of ATP-dependent calcium oscillations. Correspondingly, EGTA would increase the apoptotic index of the first stage by promoting a decrease in the calcium oscillatory rate. The fact that the second stage of apoptosis is not affected by the drugs suggests that the two stages are at least partially subject to different signaling pathways.

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.

PURPOSE: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

Further genetic and clinical insights of posterior polymorphous corneal dystrophy 3.

IMPORTANCE: Posterior polymorphous corneal dystrophy (PPCD) is a very rare disorder characterized by primary changes of the posterior corneal layers. Sequence variants in 3 genes are associated with the development of PPCD, including ZEB1 that is responsible for PPCD3. Evidence suggests at least 1 more gene remains to be identified. OBJECTIVE: To determine the molecular genetic cause of PPCD3. DESIGN: We performed extensive ophthalmological examination, including rotating Scheimpflug imaging technology and specular microscopy, and direct sequencing of the ZEB1 coding region. Comprehensive review of published PPCD3-causing variants was undertaken. SETTING: Ophthalmology department of a university hospital. PARTICIPANTS: Four Czech probands. MAIN OUTCOMES AND MEASURES: Results of ophthalmological examination and direct sequencing of the ZEB1 coding region. RESULTS: The following 2 novel frameshift mutations within ZEB1 were identified: c.2617dup in exon 8 in a 22-year-old woman, considered to be most likely de novo in origin, and c.698dup in exon 6 in a 20-year-old man. The first patient had mild changes consistent with PPCD and bilateral best-corrected visual acuity of 1.00. The corneal phenotype of the patient in the second case was more severe, with best-corrected visual acuity of 0.40 OD and 0.05 OS. Corneas of both probands were abnormally steep (keratometry readings, flat ≥ 47.4 diopters [D] and steep ≥ 49.2 D) with increased pachymetry values but no pattern indicative of keratoconus. Specular microscopy in both patients revealed reduced endothelial cell density (range, 1055/mm² to 1655/mm²). Both probands had a history of surgery for inguinal hernia; the male patient also reported hydrocele. CONCLUSIONS AND RELEVANCE: Nucleotide changes within the coding region of ZEB1 underlie the pathogenesis of PPCD in 4 of 23 Czech probands (17%). The cumulative de novo ZEB1 mutation rate is at least 14%. Possible involvement of ZEB1 sequence variants not readily identified by direct sequencing of coding regions needs to be further investigated. Our findings also have implications for patient counseling.