Peripheral ulcerative keratitis in a patient with granulomatous rosacea.

A 24-year-old woman visited our emergency department due to intermittent dull pain in the right eye, blurred vision, foreign body sensation for 3 weeks, and progressive facial rash with pustules for 3 months. She had a history of recurring skin rash on her face and extremities since early adolescence. Peripheral ulcerative keratitis (PUK) was diagnosed based on slit-lamp examination and corneal topography and then granulomatous rosacea (GR) based on clinical manifestations and skin pathology. Topical prednisolone, artificial tears, oral doxycycline, oral prednisolone, and topical clindamycin were administered. After 1 month, PUK progressed to corneal perforation probably due to eye rubbing. The corneal lesion was repaired with a glycerol-preserved corneal graft. A dermatologist prescribed oral isotretinoin for 2 months in conjunction with topical betamethasone gradually tapered for 14 months. After 34 months of follow-up, no signs of skin and ocular recurrence were noted, and the cornea graft was intact. In conclusion, PUK may present with GR, and oral isotretinoin may be an effective therapy for PUK in the setting of GR.

Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells.

Understanding the regulatory mechanisms underlying corneal epithelial cell (CEC) proliferation in vitro may provide the means to boost CEC production in cell therapy for ocular disorders. The transcription factor ΔNp63 plays a crucial role in the proliferation of CECs, but the underlying mechanisms is yet to be elucidated. TP63 and ΔNp63 are encoded by the TP63 gene via alternative promoters. We previously reported that both ΔNp63 and activating transcription factor (ATF3) are substantially expressed in cultured CECs, but the regulatory relationship between ΔNp63 and ATF3 is unknown. In the present study, we found that ΔNp63 increased ATF3 expression and ATF3 promoter activity in cultured CECs. The deletion of the p63 binding core site reduced ATF3 promoter activity. CECs overexpressing ATF3 exhibited significantly greater proliferation than control CECs. ATF3 knockdown suppressed the ΔNp63-induced increase in cell proliferation. Overexpression of ATF3 in CECs significantly elevated protein and mRNA levels of cyclin D. The protein levels of keratin 3/14, integrin ß1, and involucrin did not differ between ATF3-overexpressing CECs, ATF3-downregulated CECs, and control cells. In conclusion, our results suggest that ΔNp63 increases CEC proliferation via the ΔNp63/ATF3/CDK pathway.

Amniotic membrane transplantation in a patient with impending perforated corneal ulcer caused by Streptococcus mitis: A case report and review of literature.

BACKGROUND: Streptococcus mitis (S. mitis) is an opportunistic pathogen that can lead to severe ocular infections. In previous reports, penetrating keratoplasty (PK) was usually adopted for the treatment of persistent corneal ulcers. This report describes an unusual case of nonhealing descemetocele caused by S. mitis treated by antibiotics plus amniotic membrane transplantation (AMT). CASE SUMMARY: A 63-year-old woman presented with a right persistent corneal ulcer that she had suffered from for the past 9 mo. The culture of a corneal scraping yielded S. mitis. The right eye descemetocele decreased in diameter from 3 to 0.8 mm after the continuous administration of topical vancomycin and ceftriaxone for 2 wk. Due to the slow healing, AMT was performed. Her corneal erosion healed and gradually became clear. Her visual acuity recovered from initially counting fingers to 100/200 at the last follow-up, 67 mo after AMT. CONCLUSION: Antibiotics plus AMT may be an effective alternative treatment other than PK to promote epithelialization and to reduce inflammation in the corneas complicated by S. mitis keratitis.

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.

Refractive Changes Following Premature Posterior Capsulotomy Using Neodymium:Yttrium-Aluminum-Garnet Laser.

We aimed to determine the timing of neodymium:yttrium-aluminum-garnet (Nd:YAG) laser capsulotomy on corrected-distance visual acuity (CDVA), intraocular pressure (IOP), and spherical equivalent (SE) in patients with posterior capsular opacification (PCO). There were 59 patients with unilateral PCO and a history of Nd:YAG laser capsulotomy enrolled and further divided into the early Nd:YAG group (timing < 12 months, n = 25) and late Nd:YAG group (timing > 12 months, n = 34) depending on the elapsed months from phacoemulsification to Nd:YAG laser capsulotomy. The primary outcomes were CDVA, IOP, and SE before (immediately before Nd:YAG laser capsulotomy) and after (weeks one and four after the laser treatment). The independent t test was applied to analyze the difference in CDVA, IOP, and SE between the two groups, while the generalized estimating equation with Bonferroni adjustment was conducted to evaluate the effect of all the parameters on the change in SE with adjusted odds ratio (aOR) and 95% confidence interval (CI). The CDVA showed significant improvement in both the early Nd:YAG group (p = 0.005) and the late Nd:YAG group (p = 0.001), and hyperopic change occurred in both the early Nd:YAG group (p = 0.003) and the late Nd:YAG group (p = 0.017). The early Nd:YAG group revealed more significant hyperopic change compared with the late Nd:YAG group four weeks after Nd:YAG treatment (p < 0.001), which was still significant after multivariable analysis (aOR: 0.899, 95% CI: 0.868-0.930, p = 0.011). In addition, a deeper ACD (aOR: 0.764, 95% CI: 0.671-0.869, p = 0.019) was significantly correlated with hyperopic change. In conclusion, Nd:YAG laser capsulotomy performed within one year after cataract surgery may lead to significant hyperopic change, in which the ACD alteration affects the hyperopic shift significantly.

Corneal Endothelial Changes Following Early Capsulotomy Using Neodymium:Yttrium-Aluminum-Garnet Laser.

We aimed to survey whether the timing of neodymium:yttrium-aluminum-garnet (Nd:YAG) laser capsulotomy would alter the corneal endothelial morphology and density. A retrospective cohort study was conducted, and 48 patients with unilateral posterior capsular opacity (PCO) and Nd:YAG laser capsulotomy performance were enrolled. The participants were divided into the early Nd:YAG group (timing ≤ 12 months, n = 20) and late Nd:YAG group (timing > 12 months, n= 28) depending on elapsed months between phacoemulsification and Nd:YAG laser capsulotomy. Endothelial cell density (ECD), coefficient of variant (CV), hexagonality (HEX), and central corneal thickness (CCT) between the two groups were collected. A generalized estimate equation was conducted to evaluate the corneal endothelial parameters between the two groups with an adjusted odds ratio (aOR) and 95% confidence interval (CI). The CDVA was improved after treatment in both groups (both p < 0.001). Chronically, ECD in the early group was significantly decreased one week after treatment (2221.50 ± 327.73/mm2 vs. 2441.55 ± 321.80/mm2, p < 0.001), which recovered to 2369.95 ± 76.37/mm2 four weeks after the treatment but was still lower than the preoperative status (p < 0.001). In addition, the HEX percentage showed a significant reduction at four weeks after treatment (p = 0.028). The ECD in the early group was significantly lower than that in the late group (aOR: 0.167, 95% CI: 0.079-0.356, p = 0.003) in both week 1 (p < 0.001) and week 4 (p = 0.004) after laser treatment. In conclusion, the early application of Nd:YAG laser capsulotomy within one year after cataract surgery may be the reason for postoperative ECD decrement without known etiology.

Capturing Pluripotency and Beyond.

During the development of a multicellular organism, the specification of different cell lineages originates in a small group of pluripotent cells, the epiblasts, formed in the preimplantation embryo. The pluripotent epiblast is protected from premature differentiation until exposure to inductive cues in strictly controlled spatially and temporally organized patterns guiding fetus formation. Epiblasts cultured in vitro are embryonic stem cells (ESCs), which recapitulate the self-renewal and lineage specification properties of their endogenous counterparts. The characteristics of totipotency, although less understood than pluripotency, are becoming clearer. Recent studies have shown that a minor ESC subpopulation exhibits expanded developmental potential beyond pluripotency, displaying a characteristic reminiscent of two-cell embryo blastomeres (2CLCs). In addition, reprogramming both mouse and human ESCs in defined media can produce expanded/extended pluripotent stem cells (EPSCs) similar to but different from 2CLCs. Further, the molecular roadmaps driving the transition of various potency states have been clarified. These recent key findings will allow us to understand eutherian mammalian development by comparing the underlying differences between potency network components during development. Using the mouse as a paradigm and recent progress in human PSCs, we review the epiblast's identity acquisition during embryogenesis and their ESC counterparts regarding their pluripotent fates and beyond.

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.

Ascorbic acid ameliorates corneal endothelial dysfunction and enhances cell proliferation via the noncanonical GLUT1-ERK axis.

BACKGROUND: The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. METHODS: Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography-mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. INTERPRETATION: We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.

Accelerated Corneal Endothelial Cell Loss after Phacoemulsification in Patients with Mildly Low Endothelial Cell Density.

By evaluating preoperative endothelial cell density (ECD), ECD loss after phacoemulsification can be predicted. In this retrospective cross-sectional study, we compared outcomes of phacoemulsification with different levels of preoperative ECD. Three-hundred-and-fifty-three patients aged between 18 and 90 years received phacoemulsification at Chang Gung Memorial Hospital. Age (p = 0.003), preoperative logMAR (p = 0.048), cataract grade (p = 0.005), preoperative ECD (p < 0.001), operation time (p = 0.043), phacoemulsification time (p = 0.001), and phacoemulsification energy (p < 0.001) were significantly associated with postoperative ECD change (%). Patients were divided into three groups according to preoperative ECD levels. Level of ECD, coefficient of variation (CV), cell hexagonality (HEX), central corneal thickness (CCT), visual acuity, underlying diseases, and complications were analyzed. With regard to groups, 29, 71, and 252 patients were respectively allocated into the markedly low (group A; ECD below 1000 cells/mm2), mildly low (group B; ECD between 1000 to 2000 cells/mm2), and normal (group C; ECD above 2000 cells/mm2) ECD level groups. The highest CV (40.8 ± 13.9%; p < 0.001) and lowest HEX (58.4 ± 14.6%; p < 0.001) were found in group A. Significant ECD loss was found in group B (28.9 ± 9.2%) as compared to group A (19.9 ± 5.4%) and C (15.0 ± 12.0%) (p < 0.001). No significant differences were found with regard to changes in CV (p = 0.941), HEX (p = 0.937), CCT (p = 0.346), and logMAR (p = 0.557) among the three groups. In conclusion, preoperative ECD level could be a novel predictive value for postoperative cell loss, which was the most prominent in mildly low ECD level group. Less phacoemulsification energy, earlier surgical intervention, or novel topical medications could be suggested for patients with an ECD range from 1000 to 2000 cells/mm2.

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.

Accelerated corneal endothelial cell loss in two patients with granulomatosis with polyangiitis following phacoemulsification.

BACKGROUND: Generally, the loss rate of human endothelial cells (HCEC) in routine cataract surgery is 8.5%. When the corneal endothelial cells density (ECD) drops, the HCEC may decompensate to keep cornea dehydration which leads to corneal edema. Granulomatosis with polyangiitis (GPA) is an uncommon autoimmune disease involving multiple organs including eyes such as conjunctivitis, scleritis, uveitis, and corneal ulcer. In this study, we report two cases of GPA whose corneal ECD decreased significantly after phacoemulsification cataract surgery. CASE PRESENTATION: In the first case of 69-year-old male with GPA, the ECD dropped 39.6% (OD) four months after phacoemulsification and 38.1% (OS) six months postoperatively respectively. At the final follow-up, the residual ECD was only 55% in the right eye in the 49th month, and 56% remained in the left eye in the 39th month. In the second case of 54-year old female, left ECD dropped 63.9% at the 4th month after surgery and 69.6% ECD remained at the 15th month postoperatively while similar ECD of right eye before and after left eye surgery. CONCLUSION: Extensive preoperative ophthalmic evaluation and meticulous postoperative inflammation control should be applied to prevent severe loss of HCEC in GPA patients.

Topical Ascorbic Acid Ameliorates Oxidative Stress-Induced Corneal Endothelial Damage via Suppression of Apoptosis and Autophagic Flux Blockage.

Compromised pumping function of the corneal endothelium, due to loss of endothelial cells, results in corneal edema and subsequent visual problems. Clinically and experimentally, oxidative stress may cause corneal endothelial decompensation after phacoemulsification. Additionally, in vitro and animal studies have demonstrated the protective effects of intraoperative infusion of ascorbic acid (AA). Here, we established a paraquat-induced cell damage model, in which paraquat induced reactive oxygen species (ROS) production and apoptosis in the B4G12 and ARPE-19 cell lines. We demonstrate that oxidative stress triggered autophagic flux blockage in corneal endothelial cells and that addition of AA ameliorated such oxidative damage. We also demonstrate the downregulation of Akt phosphorylation in response to oxidative stress. Pretreatment with ascorbic acid reduced the downregulation of Akt phosphorylation, while inhibition of the PI3K/Akt pathway attenuated the protective effects of AA. Further, we establish an in vivo rabbit model of corneal endothelial damage, in which an intracameral infusion of paraquat caused corneal opacity. Administration of AA via topical application increased its concentration in the corneal stroma and reduced oxidative stress in the corneal endothelium, thereby promoting corneal clarity. Our findings indicate a perioperative strategy of topical AA administration to prevent oxidative stress-induced damage, particularly for those with vulnerable corneal endothelia.

Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin-1ß.

The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density. Few studies have investigated promoting corneal endothelial cell proliferation to increase the endothelial cell density. We hypothesize that pre-transplantation treatment of proliferative tissue-cultivated corneas may increase corneal endothelial cell density. We observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness. In this tissue culture system, we observed that lysophosphatidic acid increased the rabbit corneal endothelial cell density, number of BrdU-positive cells and improve wound healing. We also observed an indirect effect of lysophosphatidic acid on corneal endothelial cell proliferation mediated by the stimulation of interleukin-1ß secretion from stromal cells. Human corneal tissues treated with lysophosphatidic acid or interleukin-1ß contained significantly more Ki-67-positive cells than untreated group. The lysophosphatidic acid- or interleukin-1ß-treated cultured tissue remained hexagon-shaped, with ZO-1 expression and no evidence of the endothelial-mesenchymal transition. Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.

Clinical Outcomes of Penetrating Keratoplasty and Descemet Stripping Automated Endothelial Keratoplasty in Asian Population with American Corneas.

To determine the comparative efficacy and safety of penetrating keratoplasty (PK) and Descemet stripping automated endothelial keratoplasty (DSAEK) in the Asian population receiving imported donor corneas, our single-center retrospective study provides analysis supporting the transition from PK to DSAEK in the Asian population using imported American donor corneas. We analyzed 259 patients with 241 and 57 cases of PK and DSAEK respectively during 2008 to 2017 using imported corneas at Chang Gung Memorial Hospital, Linkou, Taiwan. In terms of long-term graft survival analysis, there was no difference between PK and DSAEK (log-rank p = 0.386, HR = 0.920, 95% CI: [0.641-1.380]). However, Cox proportional regression analysis revealed that corneal survival rate of DSAEK group in the first 100 days after transplantation was inferior than that of PK group (log-rank p < 0.001, HR = 2.733, 95% CI: [1.501-4.977])]. Despite the inferior survival rate, there were significantly less neovascularization and Descemet membrane folds in the DSAEK group. Importantly, the non-complication rate of DSAEK was much higher than that of PK with significant difference (PK, 25.7% vs DSAEK 42.0%, p = 0.022). Collectively, DSAEK is suggested as an alternative surgical modality in Asian patients using imported American donor corneas because of less complication, and no difference in long-term corneal graft survival rates between PK and DSAEK.

Perioperative topical ascorbic acid for the prevention of phacoemulsification-related corneal endothelial damage: Two case reports and review of literature.

BACKGROUND: The current case report describes successful phacoemulsification with the aid of perioperative topical ascorbic acid (AA) in two patients with corneal endothelial disorders to prevent postoperative corneal endothelial decompensation. CASE SUMMARY: Two eyes of two patients underwent phacoemulsification with pre-existing corneal endothelial disorders including Fuchs corneal endothelial dystrophy (Patient 1) and endotheliitis (Patient 2). Topical AA was applied to both patients at least one month before and after with a frequency of four times per day. After the surgery, both eyes improved best-corrected visual acuity (BCVA) and there was limited human corneal endothelial cell loss without signs of corneal endothelial decompensation, such as deteriorated BCVA or persistent corneal edema during the follow-up of at least two years. CONCLUSION: Perioperative administration of topical AA may be an alternative therapy to the triple procedure in patients expecting to undergo cataract surgery.

Role of dysfunctional adipocytes in cholesterol-induced nonobese metabolic syndrome.

Many studies have reported the causes of obese metabolic syndrome (MS); however, the causes of nonobese MS (NMS) remain unknown. In this study, we demonstrated that inflamed dysfunctional adipose tissue plays a crucial role in cholesterol-induced NMS. Control (C), high cholesterol (HC) and HC with 10% fructose in drinking water (HCF) diets were fed to Sprague-Dawley rats for 12 weeks. After 12 weeks, the body weights of the C- and HC-fed rats were comparable, but the weights of the HCF-fed rats were relatively low. Cholesterol caused metabolic problems such as high blood pressure, hypercholesterolemia and hypoinsulinemia. The HCF-fed rats exhibited whole-body insulin resistance with low circulating high-density lipoprotein levels. Increases in the tumor necrosis factor α level in the plasma, the number of CD68+ macrophages and the free nuclear factor-κB level in gonadal white adipose tissue (gWAT) resulted in local inflammation, which appeared as inflamed dysfunctional gWAT. Reduced superoxide dismutases (SODs) deteriorate natural antioxidant defense systems and induce reactive oxygen species in gWAT. Dysregulation of plasma levels of catecholamine, adipokines (leptin and adiponectin), hormone-sensitive lipase and perilipin in cholesterol-induced inflamed adipose tissue contributed to increased lipolysis and increased circulating nonesterified fatty acids. Cholesterol activated inflammation, lipolysis and cell death in 3T3-L1 adipocytes. Moreover, Chol-3T3-CM reduced the population of M2-type Raw264.7 macrophages, indicating that the macrophage polarization is mediated by cholesterol. Together, our findings indicate that inflamed dysfunctional adipocytes are critical in NMS, supporting the development of anti-inflammatory agents as potential therapeutic drugs for treating NMS.

Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency.

A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEFCol1a14F2A-Oct4-GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor ß (TGF-ß) in the serum by SB431542 neither affected the growth rate of MEFCol1a14F2A-Oct4-GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-ß expression was 10-fold higher in γMEFICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEFCol1a14F2A-Oct4-GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEFCol1a14F2A-Oct4-GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.

EpEX/EpCAM and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2α.

Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2α. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2α signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.