Adipose tissue dysfunction is critical to the development of type II diabetes and other metabolic diseases. While monolayer cell culture has been useful for studying fat biology, 2D culture often does not reflect the complexity of fat tissue. Animal models are also problematic in that they are expensive, time consuming, and may not completely recapitulate human biology because of species variation. To address these problems, we have developed a scaffold-free method to generate 3D adipose spheroids from primary or immortal human or mouse pre-adipocytes. Pre-adipocytes self-organize into spheroids in hanging drops and upon transfer to low attachment plates, can be maintained in long-term cultures. Upon exposure to differentiation cues, the cells mature into adipocytes, accumulating large lipid droplets that expand with time. The 3D spheroids express and secrete higher levels of adiponectin compared to 2D culture and respond to stress, either culture-related or toxin-associated, by secreting pro-inflammatory adipokines. In addition, 3D spheroids derived from brown adipose tissue (BAT) retain expression of BAT markers better than 2D cultures derived from the same tissue. Thus, this model can be used to study both the maturation of pre-adipocytes or the function of mature adipocytes in a 3D culture environment.
Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.
The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. Lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.
Neuroinflammation significantly contributes to the pathophysiology of several neurodegenerative diseases. This is also the case in glaucoma and may be a reason why many patients suffer from progressive vision loss despite maximal reduction in intraocular pressure. Pioglitazone is an agonist of the peroxisome proliferator-activated receptor gamma (PPARγ) whose pleiotrophic activities include modulation of cellular energy metabolism and reduction in inflammation. In this study we employed the DBA2/J mouse model of glaucoma with chronically elevated intraocular pressure to investigate whether oral low-dose pioglitazone treatment preserves retinal ganglion cell (RGC) survival. We then used an inducible glaucoma model in C57BL/6J mice to determine visual function, pattern electroretinographs, and tracking of optokinetic reflex. Our findings demonstrate that pioglitazone treatment does significantly protect RGCs and prevents axonal degeneration in the glaucomatous retina. Furthermore, treatment preserves and partially reverses vision loss in spite of continuously elevated intraocular pressure. These data suggest that pioglitazone may provide treatment benefits for those glaucoma patients experiencing continued vision loss.
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