11 research outputs found
ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp
The knockout (KO) of the adiponectin receptor 1 (AdipoR1) gene causes retinal degeneration. Here we report that ADIPOR1 protein is primarily found in the eye and brain with little expression in other tissues. Further analysis of AdipoR1 KO mice revealed that these animals exhibit early visual system abnormalities and are depleted of RHODOPSIN prior to pronounced photoreceptor death. A KO of AdipoR1 post-development either in photoreceptors or the retinal pigment epithelium (RPE) resulted in decreased expression of retinal proteins, establishing a role for ADIPOR1 in supporting vision in adulthood. Subsequent analysis of the Mfr
IN VITRO GENERATION OF HUMAN RETINAL GANGLION CELLS VIA DIRECT CONVERSION AND STEM CELL DIFFERENTIATION
Retinal ganglion cells (RGCs) are essential for human visual perception, as they mediate the transfer of visual information from the eye to the brain. In the event of RGC death, as happens in diseases such as glaucoma, vision is permanently lost because the mammalian central nervous system (CNS) does not regenerate. In order to find novel treatments for the RGC diseases, a number of different research approaches have been undertaken. One relatively recent approach involves the use of pluripotent stem cell (PSC) technologies. As human PSCs can be differentiated to the RGC lineage, it has become possible to utilize human RGCs for drug discovery, disease modeling, or transplantation experiments to attempt visual system recovery.
The field of PSC differentiation to RGCs has been steadily developing, but many hurdles remain. Initial reports involved co-culture of mouse stem cells with primary embryonic retinal cells or conditioned media, and yielded only small numbers of cells. Evolution of ocular stem cell biology over the past few years has greatly improved the technology for generation of three-dimensional optic vesicles that develop all of the retinal layers, including the RGC layer. Although such reports were very encouraging, these studies failed to demonstrate that RGCs could be isolated from culture and deeper profiles of the RGCs were missing. Here, we describe a novel, simplified protocol for RGC differentiation from human PSCs. We took advantage of recently developed CRISPR-Cas9 technologies to genetically engineer a stem cell reporter line for RGCs based on the BRN3B/POU4F2 gene. Using this line, we developed a fluorescence-activated cell sorting (FACS) protocol that yields highly purified RGCs, and we subsequently characterized the purified cells in terms of their expression pattern of RGC-associated genes and other cellular properties. Despite the power of FACS to provide purified populations of fluorescent RGCs, FACS-based purification schemes also have their limitations. A main limitation is the relatively slow speed and restricted throughput of FACS. In order to get around these limitations, we developed an alternative approach by genetically engineering a unique cell surface antigen driven by the BRN3B gene into stem cells, allowing us to isolate populations of pure differentiated RGCs by affinity purification in an efficient, large scale, and time saving manner that makes possible the use of human RGCs in a variety of studies including high throughput drug discovery screens. In addition to developing this new RGC purification strategy, which has implications for developing improved systems for the purification of a wide variety of different cell types, we have also optimized our initial differentiation protocol by manipulating known signaling pathways via small molecule supplementation to guide the cells toward a retinal cell fate.
In addition to developing and characterizing improved stem cell-based approaches for generating RGCs, we have also been pursuing the generation of RGCs from non-stem cell lines through trans-differentiation and direct reprogramming. Through this work, we have identified a combination of four transcription factors that can directly reprogram the human retinal pigment epithelium (RPE) cell line ARPE19 into RGC-like cells. Expression of ATOH7, BRN2, BRN3B, and MYT1 in ARPE19 cells transformed their morphology to a neuronal phenotype and induced the expression of pan-neuronal and RGC-associated genes. Moreover, when overexpressed in stem cells undergoing retinal differentiation, these factors were able to boost the percentage of cells differentiating to the RGC lineage, an effect that could be further increased by our previously identified small molecules.
Lastly, we discuss the development of a new method to enhance homology directed repair for the purpose of generating reporter lines in an easier and more routine way. Taken together, these studies provide powerful tools for the use of stem cell technology to study RGC differentiation and biology, and the mechanisms of RGC injury and cell death. Additionally, they provide the means to provide well-characterized and large supplies of RGCs for drug discovery screens and lay the groundwork for possible future cell-based therapeutic approaches for treatment of vision loss and blindness from glaucoma and other forms of optic nerve disease
Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.
Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection.
Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs
Egr2 overexpression in Schwann cells increases myelination frequency in vitro
Schwann cells are key players in peripheral nerve regeneration, and are uniquely capable of remyelinating axons in this context. Schwann cells orchestrate this process via a set of transcription factors. While it has been shown that overexpression of specific genes, e.g. Egr2, upregulates myelin-related transcripts, it remains unknown if such manipulation can functionalize the cells and enhance their myelination frequency. The ability to do so could have implications in the use of human stem cell-derived Schwann cells, where myelination is hard to achieve. After screening four candidate transcription factors (Sox10, Oct6, Brn2 and Egr2), we found that overexpression of Egr2 in rat Schwann cells co-cultured with sensory neurons enhanced myelination frequency and reduced cell proliferation. However, in a mouse model of sciatic nerve repair with cells engrafted within a nerve guide, myelination frequency in the engrafted cells was reduced upon Egr2 overexpression. Our results show that while overexpression of Egr2 can enhance the myelination frequency in vitro, it is context-dependent, potentially influenced by the microenvironment, timing of association with axons, expression level, species differences, or other factors
Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells
Abstract Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs
Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation
Single cell RNA sequencing of stem cell-derived retinal ganglion cells
We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype