A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

Low-dose minocycline mediated neuroprotection on retinal ischemia-reperfusion injury of mice.

Purpose: The aim of this study was to investigate the effect of minocycline (MC) on the survival of retinal ganglion cells (RGCs) in an ischemic-reperfusion (I/R) injury model of retinal degeneration. Methods: Retinal I/R injury was induced in the left eye of mice for 60 min by maintaining intraocular pressure at 90 mmHg. Low- or high-dose MC (20 or 100 mg/kg, respectively) was administered by intravenous injection at 5 min after the retinal ischemic insult and then administered once daily until the mice were euthanized. RGCs and microglial cells were counted using immunofluorescence staining. Functional changes in the RGCs were evaluated using electroretinography. The visual function was assessed using an optokinetic test. Results: The data demonstrated that the effect of MC was dose dependent. Low-dose MC showed protective effects, with reduced RGC loss and microglial activation, while the high-dose MC showed damage effects, with more RGC loss and microglial activation when compared with the vehicle group. The electroretinography and optokinetic test results were consistent with the morphologic observations. Conclusions: These data suggested that appropriate concentrations of MC can protect the retina against retinal ischemic-reperfusion injury, while excessive MC has detrimental effects.

Development and evaluation of a risk prediction model for diabetes mellitus type 2 patients with vision-threatening diabetic retinopathy.

Objective: This study aims to develop and evaluate a non-imaging clinical data-based nomogram for predicting the risk of vision-threatening diabetic retinopathy (VTDR) in diabetes mellitus type 2 (T2DM) patients. Methods: Based on the baseline data of the Guangdong Shaoguan Diabetes Cohort Study conducted by the Zhongshan Ophthalmic Center (ZOC) in 2019, 2294 complete data of T2DM patients were randomly divided into a training set (n=1605) and a testing set (n=689). Independent risk factors were selected through univariate and multivariate logistic regression analysis on the training dataset, and a nomogram was constructed for predicting the risk of VTDR in T2DM patients. The model was evaluated using receiver operating characteristic (ROC) curves and area under the curve (AUC) in the training and testing datasets to assess discrimination, and Hosmer-Lemeshow test and calibration curves to assess calibration. Results: The results of the multivariate logistic regression analysis showed that Age (OR = 0.954, 95% CI: 0.940-0.969, p = 0.000), BMI (OR = 0.942, 95% CI: 0.902-0.984, p = 0.007), systolic blood pressure (SBP) (OR =1.014, 95% CI: 1.007-1.022, p = 0.000), diabetes duration (10-15y: OR =3.126, 95% CI: 2.087-4.682, p = 0.000; >15y: OR =3.750, 95% CI: 2.362-5.954, p = 0.000), and glycated hemoglobin (HbA1C) (OR = 1.325, 95% CI: 1.221-1.438, p = 0.000) were independent risk factors for T2DM patients with VTDR. A nomogram was constructed using these variables. The model discrimination results showed an AUC of 0.7193 for the training set and 0.6897 for the testing set. The Hosmer-Lemeshow test results showed a high consistency between the predicted and observed probabilities for both the training set (Chi-square=2.2029, P=0.9742) and the testing set (Chi-square=7.6628, P=0.4671). Conclusion: The introduction of Age, BMI, SBP, Duration, and HbA1C as variables helps to stratify the risk of T2DM patients with VTDR.

Corrigendum: Potentials of Cellular Reprogramming as a Novel Strategy for Neuroregeneration.

[This corrects the article DOI: 10.3389/fncel.2018.00460.].

A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9.

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.

Potentials of Cellular Reprogramming as a Novel Strategy for Neuroregeneration.

Cellular reprogramming technology holds great potential for tissue repair and regeneration to replace cells that are lost due to diseases or injuries. In addition to the landmark discovery of induced pluripotent stem cells, advances in cellular reprogramming allow the direct lineage conversion of one somatic cell type to another using defined transcription factors. This direct reprogramming technology represents a rapid way to generate target cells in the laboratory, which can be used for transplantation and studies of biology and diseases. More importantly, recent work has demonstrated the exciting application of direct reprogramming to stimulate regeneration in vivo, providing an alternative approach to transplantation of donor cells. Here, we provide an overview of the underlying concept of using cellular reprogramming to convert cell fates and discuss the current advances in cellular reprogramming both in vitro and in vivo, with particular focuses on the neural and retinal systems. We also discuss the potential of in vivo reprogramming in regenerative medicine, the challenges and potential solutions to translate this technology to the clinic.