CD34dim cells identified as pluripotent stem cell-derived definitive hemogenic endothelium purified using bone morphogenetic protein 4.

Hemogenic endothelium (HE) plays a pivotal and inevitable role in haematopoiesis and can generate all blood and endothelial lineage cells in the aorta-gonad-mesonephros of mouse embryos. Whether definitive HE can prospectively isolate pure HE from human pluripotent stem cells that can spontaneously differentiate into heterogeneous cells remains unknown. Here, we identified and validated a CD34dim subpopulation with hemogenic potential. We also purified CD34 cells with a CXCR4- CD73- phenotype as a definitive HE population that generated haematopoietic stem cells and lymphocytes. The frequency of CXCR4- CD73- CD34dim was evidently increased by bone morphogenetic protein 4, and purified HE cells differentiated into haematopoietic cells with myeloid and T lymphoid lineages including Vδ2+ subset of γ/δ T cells. We developed a simple method to purify HE cells that were enriched in CD34dim cells. We uncovered an initial step in differentiating haematopoietic lineage cells that could be applied to basic and translational investigations into regenerative medicine.

Decision Tree Algorithm-Based Prediction of Vulnerability to Depressive and Anxiety Symptoms in Caregivers of Children With Glaucoma.

PURPOSE: Development and validation of a decision tree model (DTM) for prediction of mental health status in Korean caregivers of children with glaucoma. DESIGN: Cross-sectional study. METHODS: Caregivers of children younger than 18 years with diagnosed primary childhood glaucoma (n = 42), secondary childhood glaucoma (n = 51), and glaucoma suspect (GS; n = 36) were prospectively enrolled at Seoul National University Children's Hospital, Seoul, Korea. The participants completed 2 questionnaires, the 9-item Patient Health Questionnaire (PHQ-9) and the 7-item Generalized Anxiety Disorder (GAD-7) Assessment. A DTM analysis for discrimination of those with moderate-to-severe depressive (PHQ-9 score ≥10) and anxiety (GAD-7 score ≥11) symptoms was performed with recursive partitioning algorithms based on the obtained clinical, demographic, and socioeconomic data. RESULTS: The mean PHQ-9 and GAD-7 scores did not significantly differ among the 3 groups (P = .823 for PHQ-9 score; P = .730 for GAD-7 score). The DTM's balanced accuracies were 0.875 (95% CI = 0.778-0.972) for the PHQ-9 score and 0.880 (95% CI = 0.800-0.960) for theGAD-7 score. The DTM of the PHQ-9 revealed that in caregivers of children with glaucoma, depressive symptoms should be strongly suspected when (1) the child has undergone more than 2 glaucoma surgeries; or (2) the visual acuity (VA, converted to logarithm of minimum angle of resolution [logMAR]) in the better eye is worse than 0.4 if the child has had only 1 or no surgery. CONCLUSIONS: Evaluation of the number of glaucoma surgeries and VA in the better eye can be a useful decision support tool in predicting mental illness in caregivers of children with glaucoma.

Amino Acid Profiles and Biopotentiality of Hydrolysates Obtained from Comb Penshell (Atrina pectinata) Viscera Using Subcritical Water Hydrolysis.

The recovery of amino acids and other important bioactive compounds from the comb penshell (Atrina pectinata) using subcritical water hydrolysis was performed. A wide range of extraction temperatures from 140 to 290 °C was used to evaluate the release of proteins and amino acids. The amount of crude protein was the highest (36.14 ± 1.39 mg bovine serum albumin/g) at 200 °C, whereas a further increase in temperature showed the degradation of the crude protein content. The highest amount of amino acids (74.80 mg/g) was at 230 °C, indicating that the temperature range of 170-230 °C is suitable for the extraction of protein-rich compounds using subcritical water hydrolysis. Molecular weights of the peptides obtained from comb penshell viscera decreased with the increasing temperature. SDS-PAGE revealed that the molecular weight of peptides present in the hydrolysates above the 200 °C extraction temperature was ≤ 1000 Da. Radical scavenging activities were analyzed to evaluate the antioxidant activities of the hydrolysates. A. pectinata hydrolysates also showed a particularly good antihypertensive activity, proving that this raw material can be an effective source of amino acids and marine bioactive peptides.

Detection of infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in whiteleg shrimp (Penaeus vannamei) imported from Vietnam to South Korea.

In this study, whiteleg shrimp (Penaeus vannamei) imported from Vietnam were collected from South Korean markets, and examined for 2 viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV, recently classified as decapod penstyldensovirus-1), and white spot syndrome virus (WSSV). Among 58 samples, we detected IHHNV in 23 samples and WSSV in 2 samples, using polymerase chain reaction and sequencing analyses. This is the first report of IHHNV and WSSV detection in imported shrimp, suggesting that greater awareness and stricter quarantine policies regarding viruses infecting shrimp imported to South Korea are required.

Effects of dynamic oxygen concentrations on the development of mouse pre- and peri-implantation embryos using a double-channel gas supply incubator system.

OBJECTIVE: We aimed to evaluate the effects of different oxygen conditions (20% [high O2], 5% [low O2] and 5% decreased to 2% [dynamic O2]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. METHODS: The high-O2 and low-O2 groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O2 group, mouse embryos were cultured from the one-cell to the morula stage under 5% O2 for 3 days, followed by culture under 2% O2 to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. RESULTS: The blastocyst formation rate was significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group. The total cell number was significantly higher in the dynamic-O2 group than in the low-O2 and high-O2 groups. Additionally, the apoptotic index was significantly lower in the low-O2 and dynamic-O2 groups than in the high-O2 group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group. CONCLUSION: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

Prognostic Factors of Orbital Fractures with Muscle Incarceration.

BACKGROUND: Among the various signs and symptoms of orbital fractures, certain clinical findings warrant immediate surgical exploration, including gaze restriction, computed tomographic (CT) evidence of entrapment, and prolonged oculocardiac reflex. Despite proper surgical reconstruction, prolonged complications such as diplopia and gaze restriction can occur. This article evaluated the prognostic factors associated with prolonged complications of orbital fractures with muscle incarceration. METHODS: The medical records of 37 patients (37 orbits) with an orbital fracture with muscle incarceration from January 2001 to January 2015 were reviewed. The presence of Incarcerated muscle was confirmed via CT, as well as by intraoperative findings. Various factors potentially contributing to complications lasting for over 1 year after the injury were categorized and analyzed, including age, cause of injury, injury-to-operation time, operative time, fracture type, nausea, vomiting and other concomitant symptoms and injuries. RESULTS: All patients who presented with extraocular muscle limitations, positive CT findings, and/or a positive forced duction test underwent surgery. Of the 37 patients, 9 (24%) exhibited lasting complications, such as diplopia and gaze restriction. The mean follow-up period was 18.4 months (range, 1-108 months), while that of patients who experienced prolonged complications was 30.1 months (range, 13-36 months). Two factors were significantly associated with prolonged complications: injury-to-operation time and nausea/vomiting. Loss of vision, worsening of motility, and implant complication did not occur. CONCLUSIONS: Patients who present with gaze limitations, with or without other signs of a blow-out fracture, require a thorough evaluation and emergent surgery. A better prognosis is expected with a shorter injury-to-operation time and lack of nausea and vomiting at the initial presentation.

Modacrylic anion-exchange fibers for Cr(VI) removal from chromium-plating rinse water in batch and flow-through column experiments.

The aim of this study was to investigate Cr(VI) removal from chromium-plating rinse water using modacrylic anion-exchange fibers (KaracaronTM KC31). Batch experiments were performed with synthetic Cr(VI) solutions to characterize the KC31 fibers in Cr(VI) removal. Cr(VI) removal by the fibers was affected by solution pH; the Cr(VI) removal capacity was the highest at pH 2 and decreased gradually with a pH increase from 2 to 12. In regeneration and reuse experiments, the Cr(VI) removal capacity remained above 37.0 mg g-1 over five adsorption-desorption cycles, demonstrating that the fibers could be successfully regenerated with NaCl solution and reused. The maximum Cr(VI) removal capacity was determined to be 250.3 mg g-1 from the Langmuir model. In Fourier-transform infrared spectra, a Cr = O peak newly appeared at 897 cm-1 after Cr(VI) removal, whereas a Cr-O peak was detected at 772 cm-1 due to the association of Cr(VI) ions with ion-exchange sites. X-ray photoelectron spectroscopy analyses demonstrated that Cr(VI) was partially reduced to Cr(III) after the ion exchange on the surfaces of the fibers. Batch experiments with chromium-plating rinse water (Cr(VI) concentration = 1178.8 mg L-1) showed that the fibers had a Cr(VI) removal capacity of 28.1-186.4 mg g-1 under the given conditions (fiber dose = 1-10 g L-1). Column experiments (column length = 10 cm, inner diameter = 2.5 cm) were conducted to examine Cr(VI) removal from chromium-plating rinse water by the fibers under flow-through column conditions. The Cr(VI) removal capacities for the fibers at flow rates of 0.5 and 1.0 mL min-1 were 214.8 and 171.5 mg g-1, respectively. This study demonstrates that KC31 fibers are effective in the removal of Cr(VI) ions from chromium-plating rinse water.

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source.

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated ß-galactosidase (SA-ß-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFß, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-ß-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

Differential regulation of senescence and in vitro differentiation by 17ß-estradiol between mesenchymal stem cells derived from male and female mini-pigs.

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17ß-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10(-12) M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10(-12) M E2 resulted in down-regulation of ß-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.

Therapeutic potential of induced neural stem cells for spinal cord injury.

The spinal cord does not spontaneously regenerate, and treatment that ensures functional recovery after spinal cord injury (SCI) is still not available. Recently, fibroblasts have been directly converted into induced neural stem cells (iNSCs) by the forced expression defined transcription factors. Although directly converted iNSCs have been considered to be a cell source for clinical applications, their therapeutic potential has not yet been investigated. Here we show that iNSCs directly converted from mouse fibroblasts enhance the functional recovery of SCI animals. Engrafted iNSCs could differentiate into all neuronal lineages, including different subtypes of mature neurons. Furthermore, iNSC-derived neurons could form synapses with host neurons, thus enhancing the locomotor function recovery. A time course analysis of iNSC-treated SCI animals revealed that engrafted iNSCs effectively reduced the inflammatory response and apoptosis in the injured area. iNSC transplantation also promoted the active regeneration of the endogenous recipient environment in the absence of tumor formation. Therefore, our data suggest that directly converted iNSCs hold therapeutic potential for treatment of SCI and may thus represent a promising cell source for transplantation therapy in patients with SCI.

Direct conversion of mouse fibroblasts into induced neural stem cells.

Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4-5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.

Spontaneous corneal perforation in an eye with Peters' anomaly.

A premature female infant underwent her first ophthalmologic examination at the age of 4 weeks. The initial examination of the baby was requested for evaluation of a 'white spot' on the surface of her right eye. She had been hospitalized in the neonatal intensive care unit because of systemic abnormalities, such as a right clavicle fracture and microcephaly. Slit-lamp examination of the right eye showed a central corneal opacity, corneal thinning, and an iridocorneal adhesion. The lens and fundus of the right eye could not be observed. We observed no pathologic findings in the left eye. The baby's parents were informed of the high risk for spontaneous corneal perforation without external pressure. At 42 days of age, an ophthalmologic examination of the infant was again requested for evaluation of 'tears' from her right eye 3 hours previously. Examination revealed corneal perforation, iris protrusion, and a fat anterior chamber. We performed emergent conjunctival flap surgery. Three months following surgery, the patient's right eye was successfully preserved with no sign of inflammation or leakage.

Abrupt spontaneous suprachoroidal hemorrhage post-23-gauge vitrectomy during peritoneal dialysis.

Herein, we report a case of abrupt suprachoroidal hemorrhage (SCH) that developed during peritoneal dialysis in a patient with proliferative diabetic retinopathy. A 53-year-old female patient visited our clinic with blurred vision due to vitreous hemorrhage and proliferative diabetic retinopathy. Her medical history included diabetes, hypertension, chronic renal failure, and she had received scheduled peritoneal dialysis. No anticoagulant agents were used. We performed combined phacoemulsification with intraocular lens implantation and vitrectomy without any complications. Two hours later, the retina was stable and the intraocular pressure (IOP) was 11 mmHg. Four hours later, while receiving peritoneal dialysis, she abruptly developed ocular pain. Examination of her eye revealed an IOP of 38 mmHg and a SCH in the entire peripheral retina and posterior pole. At 12 hours after surgery (on the same day), the SCH was found to be further aggravated, and because a "kissing retina" was imminent, silicone oil was injected. An attempted fluid-air exchange failed because there was not enough space to fill with silicone oil due to aggravation of the SCH. Sclerotomies were performed to remove the SCH, and to create space for the silicone oil injection. Two months after surgery, the silicone oil was removed and her visual acuity was found to have improved to 20/40, but the patient died of pontine hemorrhage 1 month later. SCH can occur in vitrectomized eyes due to an increase in abdominal pressure during peritoneal dialysis, because chronic renal failure patients with diabetes and hypertension have structural vulnerabilities and vascular weaknesses due to arterial sclerosis in response to the increased blood pressure.

Intravitreal ranibizumab for acute central serous chorioretinopathy.

BACKGROUND/AIMS: To evaluate the effectiveness of intravitreal ranibizumab injection (IVRI) for acute central serous chorioretinopathy (CSC). METHODS: Patients with symptomatic CSC of less than 3 months were prospectively recruited. Patients (n = 20/group) were randomly assigned to IVRI (0.5 mg/0.05 ml) or observation and followed for 6 months. logMAR best-corrected visual acuity (BCVA), fluorescein angiography, indocyanine angiography, and central foveal thickness (CFT) were assessed at baseline and at regular follow-ups. RESULTS: All patients had increased BCVA, decreased CFT, and resolution of the neurosensory detachment. Complete resolution of neurosensory retinal detachment required more time in the observation group (13.0 ± 3.1 vs. 4.2 ± 0.9 weeks; p < 0.001). Mean BCVA and mean CFT improved significantly in both groups, but the changes were not significantly different between groups at 6 months. CONCLUSIONS: IVRI for acute CSC might hasten resolution of neurosensory detachment compared to observation alone. At 6 months, BCVA and CFT did not differ between IVRI and observation groups. Further studies are required to determine the long-term benefits of IVRI.

Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light.

High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

Conjunctival Inclusion Cysts in Long-standing Chronic Vernal Keratoconjunctivitis.

PURPOSE: To report a case of conjunctival inclusion cysts on the corneal limbus of a patient with chronic vernal keratoconjunctivitis during 16 months' follow up. METHODS: The patient was a 26 year old male without any specific history of surgery or trauma. Giant papillae, shield ulcers, and Horner-Trantas dots were detected. During the 16 month follow-up, Sodium cromoglycate eye drops and Prednisolone acetate 1% eye drops were given 3 times a day. During this period, conjunctival cysts were detected on the corneal limbus in both eyes. In spite of improvement of the corneal and conjunctival conditions, the conjunctival cysts did not seem to show any specific changes. For relief of foreign body sensation, excision of the conjunctival cysts and giant papillae of the left eye and histopathologic examination of the specimen was performed. RESULTS: On histopathological examination, the conjunctival cysts consisted of nonkeratinizing stratified epithelial cells filled with PAS-positive mucous substance. Inflammatory cells were not found in the vicinity. CONCLUSIONS: Conjunctival inclusion cysts can be seen as an atypical finding of long-standing vernal keratoconjunctivitis. Mechanical friction between the giant papillae and conjunctiva may be a factor in inducing the formation of the conjunctival cysts.

[Intussusception secondary to jejunal lymphangioma in a 13-year-old girl].

Lymphangioma is a benign tumor composed of numerous small or large thin-walled lymphatic spaces. It shows a predilection for head, neck, and axilla. Jejunal lymphangioma in children is extremely rare. We present a case of 13-year-old girl with jejuno-jejunal intussusception secondary to lymphangioma in jejunum. To our knowledge, this is the second report of jejunal lymphangioma causing intussusception in a child.