A transcriptomic signature of X chromosome overdosage in Saudi Klinefelter syndrome induced pluripotent stem cells.

Objective: The transcriptional landscape of Klinefelter syndromeduring early embryogenesis remains elusive. This study aimed to evaluate the impact of X chromosome overdosage in 47,XXY males induced pluripotent stem cells (iPSCs) obtained from patients with different genomic backgrounds and ethnicities. Design and method: We derived and characterized 15 iPSC lines from four Saudi 47,XXY KS patients and one Saudi 46,XY male. We performed a comparative transcriptional analysis using the Saudi KS-iPSCs and a cohort of European and North American KS-iPSCs. Results: We identified a panel of X-linked and autosomal genes commonly dysregulated in Saudi and European/North American KS-iPSCs vs 46,XY controls. Our findings demonstrate that seven PAR1 and nine non-PAR escape genes are consistently dysregulated and mostly display comparable transcriptional levels in both groups. Finally, we focused on genes commonly dysregulated in both iPSC cohorts and identified several gene-ontology categories highly relevant to KS physiopathology, including aberrant cardiac muscle contractility, skeletal muscle defects, abnormal synaptic transmission, and behavioral alterations. Conclusions: Our results indicate that a transcriptomic signature of X chromosome overdosage in KS is potentially attributable to a subset of X-linked genes sensitive to sex chromosome dosage and escaping X inactivation, regardless of the geographical area of origin, ethnicity, and genetic makeup.

Utilization of a nanostructured lipid carrier encapsulating pitavastatin-Pinus densiflora oil for enhancing cytotoxicity against the gingival carcinoma HGF-1 cell line.

Oral squamous cell carcinoma (OSCC) is the most common epithelial tumor of the oral cavity. Gingival tumors, a unique type of OSCC, account for 10% of these malignant tumors. The antineoplastic properties of statins, including pitavastatin (PV), and the essential oil of the Pinus densiflora leaf (Pd oil) have been adequately reported. The goal of this investigation was to develop nanostructured lipid carriers (NLCs) containing PV combined with Pd oil and to determine their cytotoxicity against the cell line of human gingival fibroblasts (HGF-1). A central composite quadratic design was adopted to optimize the nanocarriers. The particle size and stability index of the nano-formulations were measured to evaluate various characteristics. TEM analysis, the entrapment efficiency, dissolution efficiency, and the cytotoxic efficiency of the optimized PV-loaded nanostructured lipid carrier drug delivery system (PV-Pd-NLCs) were evaluated. Then, the optimal PV-Pd-NLCs was incorporated into a Carbopol 940® gel base and tested for its rheological features and its properties of release and cell viability. The optimized NLCs had a particle size of 98 nm and a stability index of 89%. The gel containing optimum PV-Pd-NLCs had reasonable dissolution efficiency and acceptable rheological behavior and acquired the best cytotoxic activity against HGF-1 cell line among all the formulations developed for the study. The in vitro cell viability studies revealed a synergistic effect between PV and Pd oil in the treatment of gingival cancer. These findings illustrated that the gel containing PV-Pd-NLCs could be beneficial in the local treatment of gingival cancer.

Design and Development of Neomycin Sulfate Gel Loaded with Solid Lipid Nanoparticles for Buccal Mucosal Wound Healing.

Drug administration to the wound site is a potential method for wound healing. The drug retention duration should be extended, and drug permeability through the buccal mucosal layer should be regulated. Oral wounds can be caused by inflammation, ulcers, trauma, or pathological lesions; if these wounds are not treated properly, they can lead to pain, infection, and subsequent undesirable scarring. This study aimed to develop Kolliphor-407 P-based gel containing neomycin sulfate (NES) loaded in solid lipid nanoparticles (SLNs) and enhance the antimicrobial activity. By considering lipid concentrations and achieving the lowest particle size (Y1) and maximum entrapment (EE-Y2) effectiveness, the formulation of NES-SLN was optimized using the Box-Behnken design. For the selected responses, 17 runs were formulated (as anticipated by the Design-Expert software) and evaluated accordingly. The optimized formulation could achieve a particle size of 196.25 and EE of 89.27% and was further utilized to prepare the gel formulation. The NES-SLN-G formula was discovered to have a smooth, homogeneous structure and good mechanical and rheological properties. After 24 h of treatment, NES-SLN-G showed a regulated in vitro drug release pattern, excellent ex vivo permeability, and increased in vitro antibacterial activity. These findings indicate the potential application of NES-SLN-loaded gels as a promising formulation for buccal mucosal wound healing.

Integrative global co-expression analysis identifies key microRNA-target gene networks as key blood biomarkers for obesity.

BACKGROUND: Obesity is associated with the quantitative changes in miRNAs and their target genes. However, the molecular basis of their dysregulation and expression status correlations is incompletely understood. Therefore, this study aims to examine the shared differentially expressed miRNAs and their target genes between blood and adipose tissues of obese individuals to identify potential blood-based biomarkers. METHODS: In this study, 3 gene expression datasets (two mRNA and one miRNA), generated from blood and adipose tissues of 68 obese and 39 lean individuals, were analyzed by a series of robust computational concepts, like protein interactome mapping, functional enrichment of biological pathways and construction of miRNA-mRNA and transcription factor gene networks. RESULTS: The comparison of blood versus tissue datasets has revealed the shared differential expression of 210 genes (59.5% upregulated) involved in lipid metabolism and inflammatory reactions. The blood miRNA (GSE25470) analysis has identified 79 differentially expressed miRNAs (71% downregulated). The miRNA-target gene scan identified regulation of 30 shared genes by 22miRNAs. The gene network analysis has identified the inverse expression correlation between 8 target genes (TP53, DYSF, GAB2, GFRA2, NACC2, FAM53C, JNK and GAB2) and 3 key miRNAs (hsa-mir-940, hsa-mir-765, hsa-mir-612), which are further regulated by 24 key transcription factors. CONCLUSIONS: This study identifies potential obesity related blood biomarkers from large-scale gene expression data by computational miRNA-target gene interactome and transcription factor network construction methods.

Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived retinal organoids.

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-phenyl]glycine1,1-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.

Differentiation of Retinal Organoids from Human Pluripotent Stem Cells.

This unit describes a protocol for generating retinal organoids that contain all major retinal cell types and are responsive to light from human pluripotent stem cells (hPSCs). hPSCs are differentiated in 96-well plates to allow large-scale production of organoids that could be used for multiple applications, including study of human retinal development, disease modeling, and compound screening. The differentiation approach is based on the knowledge that insulin-like growth factor 1 signaling together with retinoic acid and triiodothyronine is important for retinal development. After 22 weeks in culture, the organoids form a thick layer of neuroepithelium containing photoreceptors and bipolar, horizontal, amacrine, Müller, and retinal ganglion cells. Differentiation progress can be tracked by morphological observations and protein localization, as detected with immunocytochemistry. © 2019 by John Wiley & Sons, Inc.

Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences.

A major goal in the stem cell field is to generate tissues that can be utilized as a universal tool for in vitro models of development and disease, drug development, or as a resource for patients suffering from disease or injury. Great efforts are being made to differentiate human pluripotent stem cells in vitro toward retinal tissue, which is akin to native human retina in its cytoarchitecture and function, yet the numerous existing retinal induction protocols remain variable in their efficiency and do not routinely produce morphologically or functionally mature photoreceptors. Herein, we determine the impact that the method of embryoid body (EB) formation and maintenance as well as cell line background has on retinal organoid differentiation from human embryonic stem cells and human induced pluripotent stem cells. Our data indicate that cell line-specific differences dominate the variables that underline the differentiation efficiency in the early stages of differentiation. In contrast, the EB generation method and maintenance conditions determine the later differentiation and maturation of retinal organoids. Of the latter, the mechanical method of EB generation under static conditions, accompanied by media supplementation with Y27632 for the first 48 hours of differentiation, results in the most consistent formation of laminated retinal neuroepithelium containing mature and electrophysiologically responsive photoreceptors. Collectively, our data provide substantive evidence for stage-specific differences in the ability to give rise to laminated retinae, which is determined by cell line-specific differences in the early stages of differentiation and EB generation/organoid maintenance methods at later stages.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Laminin γ3 plays an important role in retinal lamination, photoreceptor organisation and ganglion cell differentiation.

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, ß1, ß2 and γ1 to be predominantly expressed in Bruch's membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Extracellular matrix component expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. STATEMENT OF SIGNIFICANCE: The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation.

Cellular regeneration strategies for macular degeneration: past, present and future.

Despite considerable effort and significant therapeutic advances, age-related macular degeneration (AMD) remains the commonest cause of blindness in the developed world. Progressive late-stage AMD with outer retinal degeneration currently has no proven treatment. There has been significant interest in the possibility that cellular treatments may slow or reverse visual loss in AMD. A number of modes of action have been suggested, including cell replacement and rescue, as well as immune modulation to delay the neurodegenerative process. Their appeal in this enigmatic disease relate to their generic, non-pathway-specific effects. The outer retina in particular has been at the forefront of developments in cellular regenerative therapies being surgically accessible, easily observable, as well as having a relatively simple architecture. Both the retinal pigment epithelium (RPE) and photoreceptors have been considered for replacement therapies as both sheets and cell suspensions. Studies using autologous RPE, and to a lesser extent, foetal retina, have shown proof of principle. A wide variety of cell sources have been proposed with pluripotent stem cell-derived cells currently holding the centre stage. Recent early-phase trials using these cells for RPE replacement have met safety endpoints and hinted at possible efficacy. Animal studies have confirmed the promise that photoreceptor replacement, even in a completely degenerated outer retina may restore some vision. Many challenges, however, remain, not least of which include avoiding immune rejection, ensuring long-term cellular survival and maximising effect. This review provides an overview of progress made, ongoing studies and challenges ahead.