Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing

CRISPR/Cas has opened the prospect of direct gene correction therapy for someinherited retinal diseases. Previous work has demonstrated the utility of adenoassociatedvirus (AAV) mediated delivery to retinal cells in vivo; however, with theexpanding repertoire of CRISPR/Cas endonucleases, it is not clear which of theseare most efficacious for retinal editing in vivo. We sought to compare CRISPR/Casendonuclease activity using both single and dual AAV delivery strategies for gene editingin retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNAwere generated and validated in YFP-expressing HEK293A cell by flow cytometry andthe T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA wasthen packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally intoCMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiencythan SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editingwas found between single and dual CRISPR/SaCas9 vector systems. With a markedreduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to havethe highest knockout efficacy among all investigated endonucleases. We demonstratethat the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the mostefficient gene modification in neurosensory retinal cells in vivo

Functional properties of a Brazilian derived mouse embryonic stem cell line

Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos. Since ion channel modulation has been reported to interfere with both growth and differentiation process in mouse and human ESC it is important to characterize the electrophysiological properties of newly generated mESC and compare them to other lines. In this work, we studied the intercellular communication by way of gap junctions in a Brazilian derived mESC (USP-1, generated by Dr. Lygia Pereira's group) and characterized its electrophysiological properties. We used immunofluorescence and RT-PCR to reveal the presence of connexin 43 (Cx43), pluripotency markers and ion channels. Using a co-culture of neonatal mouse cardiomyocytes with mESC, where the heart cells expressed the enhanced Green Fluorescent Protein, we performed dye injections to assess functional coupling between the two cell types observing dye diffusion. The patch-clamp study showed outward currents identified as two types of potassium currents, transient outward potassium current (Ito) and delayed rectifier outward potassium current (Iks), by use of specific drug blockage. Calcium or sodium currents in undifferentiated mESC were not identified. We conclude that USP-1 mESC has functional Cx43 channels establishing intercellular communication among themselves and with cardiomyocytes and has a similar electrophysiological profile compared to other mESC cell lines

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors

Retinal Transcriptome and Cellular Landscape in Relation to the Progression of Diabetic Retinopathy

PURPOSE: Previous studies that identify putative genes associated with diabetic retinopathy are only focusing on specific clinical stages, thus resulting genes are not necessarily reflective of disease progression. This study identified genes associated with the severity level of diabetic retinopathy using the likelihood-ratio test (LRT) and ordinal logistic regression (OLR) model, as well as to profile immune and retinal cell landscape in progressive diabetic retinopathy using a machine learning deconvolution approach. METHODS: This study used a published transcriptomic dataset (GSE160306) from macular regions of donors with different degrees of diabetic retinopathy (10 healthy controls, 10 cases of diabetes, 9 cases of nonproliferative diabetic retinopathy, and 10 cases of proliferative diabetic retinopathy or combined with diabetic macular edema). LRT and OLR models were applied to identify severity-associated genes. In addition, CIBERSORTx was used to estimate proportional changes of immune and retinal cells in progressive diabetic retinopathy. RESULTS: By controlling for gender and age using LRT and OLR, 50 genes were identified to be significantly increased in expression with the severity of diabetic retinopathy. Functional enrichment analyses suggested these severity-associated genes are related to inflammation and immune responses. CCND1 and FCGR2B are further identified as key regulators to interact with many other severity-associated genes and are crucial to inflammation. Deconvolution analyses demonstrated that the proportions of memory B cells, M2 macrophages, and Müller glia were significantly increased with the progression of diabetic retinopathy. CONCLUSIONS: These findings demonstrate that deep analyses of transcriptomic data can advance our understanding of progressive ocular diseases, such as diabetic retinopathy, by applying LRT and OLR models as well as bulk gene expression deconvolution

Repression of Global Protein Synthesis by Eif1a-Like Genes That Are Expressed Specifically in the Two-Cell Embryos and the Transient Zscan4-Positive State of Embryonic Stem Cells

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state

Differentiation of Retinal Glial Cells From Human Embryonic Stem Cells by Promoting the Notch Signaling Pathway

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells