Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases such as Stargardt's disease, age‐related macular degeneration, and retinitis pigmentosa. The ...final impact of all retinal dystrophies is the loss of photoreceptors; hence, there is a pressing need for research into replacement. Seminal work has shown that a simple three‐dimensional culture system enables differentiation of human pluripotent stem cells to retinal organoids containing large numbers of photoreceptors developing alongside retinal neurons and Müller glia cells in a laminated structure that resembles the native retina. Despite these promising developments, current protocols show different efficiencies across pluripotent stem cells and result in retinal organoids with a mixture of photoreceptor cells at varying maturation states, along with nonphotoreceptor cell types. In this study, we investigated the impact of stage‐specific addition of retinoic acid (RA), 9‐cis‐retinal, 11‐cis‐retinal, levodopa (l‐DOPA), triiodothyronine (T3), and γ‐secretase inhibitor ((2S)‐N‐(3,5‐Difluorophenyl)acetyl‐l‐alanyl‐2‐phenylglycine1,1‐dimethylethyl ester2L DAPT) in the generation of cone and rod photoreceptors. Our results indicate that addition of RA + T3 during days 90 to 120 of differentiation enhanced the generation of rod and S‐cone photoreceptor formation, while the combined addition of DAPT from days 28 to 42 with RA during days 30 to 120 of differentiation led to enhanced generation of L/M‐cones at the expense of rods. l‐DOPA when added together with RA during days 90 to 120 of differentiation also promoted the emergence of S‐cones at the expense of rod photoreceptors. Collectively, these data represent an advance in our ability to direct generation of rod and cone photoreceptors in vitro.
Schematic summary of our results showing that the addition of retinoic acid + T3 (days 90‐120) led to enhanced generation of rod and S‐cone photoreceptors while combined addition of retinoic acid + Levodopa, at the same time window, promoted only the S‐cone formation. The supplementation of retinoic acid + DAPT (days 30‐120 for RA+ days 28‐42 for DAPT) resulted in an increased L/M‐cone photoreceptor generation.
Yucca aloifolia L. fruit (Yucca or Spanish bayonet, family Asparagaceae) is recognized for its purplish red color reflecting its anthocyanin content, which has a powerful antioxidant activity. This ...study aimed to investigate yucca (YA) fruit extract's protective effect on Parkinson's disease (PD). In vitro study, the anti-inflammatory activity of yucca fruit extracts was explored by measuring tumor necrosis factor receptor 2 (TNF-R2) and nuclear factor kappa B (NF-KB) to choose the most effective extract. Afterward, a detailed in vivo investigation of the protective effect of the most active extract on rotenone-induced PD was performed on male albino Wister rats. First, the safety of the extract in two different doses (50 and 100 mg/kg in 0.9% saline orally) was confirmed by a toxicological study. The rats were divided into four groups: 1) normal control (NC); 2) rotenone group; and third and fourth groups received 50 and 100 mg/kg yucca extract, respectively. The neurobehavioral and locomotor activities of the rats were tested by rotarod, open field, and forced swim tests. Striatal dopamine, renal and liver functions, and oxidative stress markers were assessed. Western blot analysis of brain tissue samples was performed for p-AMPK, Wnt3a, and β-catenin. Histopathological examination of striatal tissue samples was performed by light and electron microscopy (EM). The metabolites of the active extract were characterized using high-resolution LC-MS/MS, and the results showed the prevalence of anthocyanins, saponins, phenolics, and choline. Biochemical and histopathological tests revealed a dose-dependent improvement with oral Yucca extract. The current study suggests a possible neuroprotective effect of the acidified 50% ethanol extract (YA-C) of the edible Yucca fruit, making it a promising therapeutic target for PD.
Destruction of the limbus and depletion of limbal stem cells (LSCs), the adult progenitors of the corneal epithelium, leads to limbal stem cell deficiency (LSCD). LSCD is a rare, progressive ocular ...surface disorder which results in conjunctivalisation and neovascularisation of the corneal surface. Many strategies have been used in the treatment of LSCD, the common goal of which is to regenerate a self-renewing, transparent, and uniform epithelium on the corneal surface. The development of these techniques has frequently resulted from collaboration between stem cell translational scientists and ophthalmologists. Direct transplantation of autologous or allogeneic limbal tissue from a healthy donor eye is regarded by many as the technique of choice. Expansion of harvested LSCs in vitro allows smaller biopsies to be taken from the donor eye and is considered safer and more acceptable to patients. This technique may be utilised in unilateral cases (autologous) or bilateral cases (living related donor). Recently developed, simple limbal epithelial transplant (SLET) can be performed with equally small biopsies but does not require in vitro cell culture facilities. In the case of bilateral LSCD, where autologous limbal tissue is not available, autologous oral mucosa epithelium can be expanded in vitro and transplanted to the diseased eye. Data on long-term outcomes (over 5 years of follow-up) for many of these procedures is needed, and it remains unclear how they produce a self-renewing epithelium without recreating the vital stem cell niche. Bioengineering techniques offer the ability to re-create the physical characteristics of the stem cell niche, while induced pluripotent stem cells offer an unlimited supply of autologous LSCs. In vivo confocal microscopy and anterior segment OCT will complement impression cytology in the diagnosis, staging, and follow-up of LSCD. In this review we analyse recent advances in the pathology, diagnosis, and treatment of LSCD.
Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only ...indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell‐based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony‐forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real‐time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti‐NGF antibody showed decreased colony‐forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony‐forming efficiency, and a maintainer of the LSC phenotype. Stem Cells 2019;37:139–149
Limbal stem cells (LSCs) harvested from human cadaveric corneoscleral rims show increased expression of putative LSC markers (ABCG2 and ΔNp63α) and greater life spans in vitro when cultured in medium supplemented with recombinant human nerve growth factor (NGF). Those cultured with NGF‐blocking antibodies show reduced expression of putative LSC markers (ABCG2 and C/EBPδ), decreased colony‐forming efficiency and proliferation.
Stem cell‐derived retinal organoids offer the opportunity to cure retinal degeneration of wide‐ranging etiology either through the study of in vitro models or the generation of tissue for ...transplantation. However, despite much work in animals and several human pilot studies, satisfactory therapies have not been developed. Two major challenges for retinal regenerative medicine are (a) physical cell‐cell interactions, which are critical to graft function, are not formed and (b) the host environment does not provide suitable queues for development. Several strategies offer to improve the delivery, integration, maturation, and functionality of cell transplantation. These include minimally invasive delivery, biocompatible material vehicles, retinal cell sheets, and optogenetics. Optimizing several variables in animal models is practically difficult, limited by anatomical and disease pathology which is often different to humans, and faces regulatory and ethical challenges. High‐throughput methods are needed to experimentally optimize these variables. Retinal organoids will be important to the success of these models. In their current state, they do not incorporate a representative retinal pigment epithelium (RPE)‐photoreceptor interface nor vascular elements, which influence the neural retina phenotype directly and are known to be dysfunctional in common retinal diseases such as age‐related macular degeneration. Advanced coculture techniques, which emulate the RPE‐photoreceptor and RPE‐Bruch's‐choriocapillaris interactions, can incorporate disease‐specific, human retinal organoids and overcome these drawbacks. Herein, we review retinal coculture models of the neural retina, RPE, and choriocapillaris. We delineate the scientific need for such systems in the study of retinal organogenesis, disease modeling, and the optimization of regenerative cell therapies for retinal degeneration.
Neural retina, retinal pigment epithelium, and choriocapillaris are highly interdependent and therefore coculture techniques are required to understand normal development and disease. Furthermore, coculture techniques will enable the optimization of regenerative therapies and the development of transplantable multitissue sheets.
Thais savignyi Deshayes (Muricidae) is widely distributed in the Red Sea. Its abundance and the history of Muricidae in traditional medicine make it a tempting target for investigation.
To ...investigate the chemical profile and biological activities of T. savignyi tissue extracts.
Methanol, ethanol, acetone, and ethyl acetate extracts from T. savignyi tissue were compared in their antioxidant by total antioxidant capacity, DPPH free radical scavenging, and total phenolic content. In addition, the antimicrobial, and antibiofilm properties (at 250 µg/mL) of the extracts were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans. The antioxidant extract with greatest activity was assessed for cytotoxicity (range 0.4-100 µg/mL) against 3 human cancer cell lines (UO-31, A549 and A431), and its chemical composition was investigated using GC-MS. Moreover, docking simulation was performed to predict its constituents' binding modes/scores to the active sites of thymidylate kinase.
The ethyl acetate extract (Ts-EtOAc) showed the highest total antioxidant capacity (551.33 mg AAE/g dry weight), total phenolics (254.46 mg GAE/g dry weight), and DPPH scavenging (IC
50
= 24.0 µg/mL). Ts-EtOAc exhibited strong antibacterial (MIC: 3.9 µg/mL against K. pneumoniae), antibiofilm (MIC: 7.81 µg/mL against S. aureus), and antifungal (MIC: 3.9 µg/mL against C. albicans) activities and considerable cytotoxicity against cancer cells (UO-31: IC
50
= 19.96 ± 0.93, A549: IC
50
= 25.04 ± 1.15 μg/mL). GC-MS identified multiple bioactive metabolites in Ts-EtOAc extract belonging to miscellaneous chemical classes. Molecular docking studies revealed that the constituents of Ts-EtOAc have antibacterial potential.
T. savignyi extract has considerable antimicrobial and cytotoxic activities. Further studies are needed to isolate the active constituents of this snail for comprehensive drug discovery tests.
Acute appendicitis is a common surgical emergency; however, its misdiagnosis resulting in negative appendicectomy is not uncommon. Novel diagnostic methods will help reduce the rate of negative ...appendicectomy. We hypothesise that the neutrophil-lymphocyte ratio (NLR) will increase upon peritoneal involvement by inflammation, as it does in severe disease.
We conducted a retrospective analysis on prospectively collected data for all emergency appendicectomy patients during the study period. We studied blood results at the time of presentation, and histology of the removed appendices. Receiver Operating Characteristics were calculated to classify histologically normal versus inflamed appendices and moderate versus severe disease. Moderate disease was that confined to the sub-serosal layers, while severe disease involved the serosa and beyond.
A total of 372 patients underwent emergency appendicectomy, 254 (78.4%) of which subsequently had acute appendicitis on histology. Sixty-five (25.6%) and 189 (74.4%) patients had moderate and severe disease, respectively. The median age was 27 years (range 16-84). In diagnosing acute appendicitis, the cut-off value of the NLR was 4.2, while the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 79.5%, 67.0%, 89.8%, and 47.5%, respectively. In identifying the moderate versus severe disease, the cut-off value was 9.7, while the sensitivity, specificity, PPV and NPV were 71.4%, 53.8%, 85.4% and 43.7%, respectively.
Inflammatory markers are useful adjuncts to history, examination, and radiological investigations in making the diagnosis of appendicitis. However, due to low sensitivity and specificity, they cannot be used alone. Calculation of the NLR has no additional benefit over the neutrophil count in diagnosis, or in distinguishing moderate and severe disease.
Limbal stem cell (LSC) deficiency causes progressive loss of vision but may be treated by transplant of autologous LSCs. Cryopreservation has the potential to indefinitely extend the lifespan of LSCs ...allowing re-transplant in case of graft failure. In this study, we aimed to identify the optimal cryoprotectant and cryoprotectant concentration for LSC cultures. Suspension cultures derived from cadaveric corneoscleral rims were cooled to 4 °C with Me2SO, propylene glycol or ethylene glycol at a concentration of 5%, 10% or 15%. Cell tolerance was measured in terms of membrane integrity, colony-forming efficiency and alamarBlue® reduction. Increasing cryoprotectant concentration above 5% reduced membrane integrity, metabolism and colony-forming efficiency. Cryoprotectant choice did not significantly influence these characteristics. Cells demonstrating Side Population were maintained after cryopreservation with 5% propylene glycol in vapour phase liquid nitrogen for 1 week, indicating that cryopreservation of LSCs with relatively low cryoprotectant concentration (5%) has promise in low-temperature eye banking.
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