Generating minicorneal organoids from human induced pluripotent stem cells.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.

Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1-/- iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines. Two of the mutant lines are compound heterozygous, with different in-del mutations in each of their alleles, while the third mutant line is homozygous, with identical edits in both alleles. All lines maintained their stemness, pluripotency, formed embryoid bodies with cell types of all three lineages, displayed a normal karyotype and lost RB1 expression.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3.

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation of an induced pluripotent stem cell line (LVPEIi002-A) with heterozygous RB1 mutation using peri-orbital fat derived mesenchymal cells of a patient with inherited retinoblastoma.

Retinoblastoma is a pediatric intraocular cancer caused by biallelic inactivation of RB1 gene in retinal progenitor cells. Here, we report the generation of a patient-specific induced pluripotent stem cell (iPSC) line (LVPEIi002-A) from a patient diagnosed with retinoblastoma and showing familial inheritance of a nonsense mutation (c.1735C > T) within exon 18 of one of the two alleles. This RB1+/- iPSC line, LVPEIi002-A was generated by reprogramming the peri-orbital fat tissue derived mesenchymal cells and was stably expanded and characterized. It maintains the stemness, pluripotency, normal karyotype, and forms embryoid bodies comprising of all three lineage committed progenitor cells.

Generation and characterization of a Stargardt's disease-specific induced pluripotent stem cell line (LVPEIi008-A) with a homozygous nonsense mutation in exon 44 of ABCA4.

The Stargardt's Disease, Type 1 (STGD1) is associated with the loss of function mutations in ABCA4. This gene codes for a retina-specific, ATP-binding cassette (ABC) family transporter, involved in the transport of the key visual cycle intermediate, all-trans-retinaldehyde (atRAL), across the photoreceptor cell membranes. Here, we report the establishment of a patient-specific, iPSC line (LVPEIi008-A), that carries a homozygous nonsense mutation at (c.6088C > T) position, within exon 44 of ABCA4. The patient-specific skin fibroblasts were reprogrammed using episomal plasmids and the stably expanding iPSC line expressed the key stemness and pluripotency markers, maintained its chromosomal integrity and tested negative for mycoplasma.

Generation of two induced pluripotent stem cell lines (LVPEIi007-B, LVPEIi008-B) from patients harboring homozygous mutation in ABCA4 (c.6088C>T) using non-integrative Sendai virus-based approach.

Mutations in ABCA4 gene leads to the most common form of an inherited retinal disease namely, the Stargardt disease, type 1. Here, we report the generation of two different patient-specific induced pluripotent stem cell lines (LVPEIi007-B and LVPEIi008-B), carrying an identical homozygous mutation, (c.6088C>T) within the exon 44 of ABCA4 gene. These lines were generated by the reprogramming of patient-specific dermal fibroblasts, using the integration-free, Sendai viral vectors. Both lines were stably expanded and expressed the stemness and pluripotency markers, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation of two induced pluripotent stem cell lines (LVPEIi004-A and LVPEIi005-A) from probands with Leber Congenital Amaurosis 2 (LCA2) and harboring mutations in RPE65.

Leber Congenital Amaurosis 2 is an early onset retinal dystrophy that occurs due to mutation in RPE65 gene. Here, we report the generation of two patient specific induced pluripotent stem cell lines harboring nonsense mutations in exon 7 (c.646A > T) and exon 9 (c.992G > A) of RPE65 gene, respectively, which leads to premature translational termination and formation of defective protein. These lines were generated by the reprogramming of human dermal fibroblast cells using integration-free, episomal constructs expressing stemness genes. The stable lines maintained a normal karyotype, expressed the key stemness factors, underwent trilineage differentiation, and maintained their genetic identity and genomic integrity.

Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

PURPOSE: To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. DESIGN: Laboratory investigation. METHODS: Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. RESULTS: The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. CONCLUSIONS: The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities.

CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Gene therapy in retinal diseases: A review.

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna-the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.

Expression of a novel brain specific isoform of C3G is regulated during development.

Mice lacking C3G (RapGEF1), a ubiquitously expressed protein essential for neuronal differentiation, show multiple defects in brain development. Function of C3G in neurogenesis is poorly defined. Here, we identify brain specific expression of a novel C3G isoform in mice and humans. This isoform has an insert in the Crk-binding region, generating a polypeptide of 175 kDa, unlike the previously known 140 kDa form expressed in all other tissues. In the adult mouse brain, C3G expression is seen in neurons, but was not detectable in GFAP-positive cells. C3G levels were high in the CA3 region of hippocampus and in mitral cells of olfactory bulb. Neural progenitor cells positive for Doublecortin and Nestin, show expression of C3G. During development, C3G is expressed in precursor cells prior to their differentiation into mature neurons or astrocytes. The 175 kDa as well as 140 kDa forms are seen in embryonic mouse brain, while only the 175 kDa variant is seen in post-natal brain. Human cerebral organoids generated from induced pluripotent stem cells predominantly expressed the 140 kDa polypeptides, and the 175 kDa isoform appeared upon maturation. This study describes developmental regulation and neuronal expression of a brain specific isoform of C3G, a molecule essential for normal development of the mammalian brain.

Learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model: Implications for future clinical trials.

Purpose: The sub-retinal injections are not very commonly performed procedures in vitreoretina, but form a crucial step in any cell replacement therapy for retinal diseases. The purpose of this study is to describe the learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model and its implications in future clinical trials. Methods: This is an in-vivo retrospective animal study using Wistar rats. All ARVO guidelines regarding animal handling were followed. After anesthetization, aspectic preparation and dilating the pupils with 1% tropicamide eye drops, subretinal injection of 10 µl saline was done via a limbal entry. Data recorded included time taken for the procedure, success of injection, associated complications, post-operative infections and complications. The rats were followed up for 1 month post procedure. A trend analysis was done for the above factors to look for improvement in ease of procedure, reduction in procedure time and reduction in complications for the clinician using a novel objective scale. Results: About 20 eyes were studied. Mean weight of the rats was 188 ± 12.82 gram. Mean time taken for the procedure was 14.1 ± 5.07 minutes. There was a significant inverse co-relation between the serial number of the eye and time taken for the procedure (r = -0.89, P < 0.0001). Comparative complications noted between the first ten and the last ten eyes were: conjunctival tear 30% versus 10% (P = 0.27), lens touch 50% versus 10% (P = 0.05), subretinal hemorrhage 40% versus 0% (P = 0.13), vitreous loss 30% versus 0% (P = 0.06). The successful subretinal injection without intraocular complications was achieved in 40% versus 90% (P = 0.02). There was a significant co-relation between the serial number of the eye and ease of the procedure (r = 0.87, P < 0.0001). Post operatively none of the eyes had any infection. Six eyes (12%) developed cataract and 3 eyes (6%) had non-resolving retinal detachment at the last examination visit. Conclusion: Subretinal injections in rats have a definite learning curve even for a trained vitreoretinal surgeon. This should be accounted for and resources allocated accordingly to achieve good technical comfort and negate confounding by the surgeon factor in the results of future clinical trials.

Sequestration of pRb by cyclin D3 causes intranuclear reorganization of lamin A/C during muscle cell differentiation.

The A-type lamins that localize in nuclear domains termed lamin speckles are reorganized and antigenically masked specifically during myoblast differentiation. This rearrangement was observed to be linked to the myogenic program as lamin speckles, stained with monoclonal antibody (mAb) LA-2H10, were reorganized in MyoD-transfected fibroblasts induced to transdifferentiate to muscle cells. In C2C12 myoblasts, speckles were reorganized early during differentiation in cyclin D3-expressing cells. Ectopic cyclin D3 induced lamin reorganization in C2C12 myoblasts but not in other cell types. Experiments with adenovirus E1A protein that can bind to and segregate the retinoblastoma protein (pRb) indicated that pRb was essential for the cyclin D3-mediated reorganization of lamin speckles. Cyclin D3-expressing myoblasts displayed site-specific reduction of pRb phosphorylation. Furthermore, disruption of lamin structures by overexpression of lamins inhibited expression of the muscle regulatory factor myogenin. Our results suggest that the reorganization of internal lamins in muscle cells is mediated by key regulators of the muscle differentiation program.

Therapeutic avenues for hereditary forms of retinal blindness.

Hereditary retinal diseases, known as retinal degenerations or dystrophies, are a large group of inherited eye disorders resulting in irreversible visual loss and blindness. They develop due to mutations in one or more genes that lead to the death of the retinal photoreceptor cells. Till date, mutations in over 200 genes are known to be associated with all different forms of retinal disorders. The enormous genetic heterogeneity of this group of diseases has posedmany challenges in understanding the mechanisms of disease and in developing suitable therapies. Therapeutic avenues that are being investigated for these disorders include gene therapy to replace the defective gene, treatment with neurotrophic factors to stimulate the growth of photoreceptors, cell replacement therapy, and prosthetic devices that can capture light and transmit electrical signals through retinal neurons to the brain. Several of these are in process of human trials in patients, and have shown safety and efficacy of the treatment. A combination of approaches that involve both gene replacement and cell replacement may be required for optimum benefit.

Surgical Management of Bilateral Limbal Stem Cell Deficiency.

Limbal stem cell deficiency (LSCD) is now established as a distinct entity with a spectrum of clinical manifestations. Bilateral LSCD presents a unique set of challenges to the clinician dealing with ocular surface disease, due to the underlying causes, clinical presentation, and adnexal status, as well as lack of a source of autologous limbal stem cells. Various surgical modalities have been described to achieve visual rehabilitation in patients with bilateral LSCD. These can primarily be divided into cell-based therapies and implantation of keratoprostheses. In this review, the surgical options for management of bilateral LSCD, including autologous and allogeneic cell-based therapies and different types of keratoprostheses are described and classified. The indications, prerequisites, technique, results and complications of each modality are discussed. Based on the status of the ocular surface, an algorithm for choosing appropriate surgical management for vision restoration in bilateral LSCD has been proposed.

Enriched Cultures of Retinal Cells From BJNhem20 Human Embryonic Stem Cell Line of Indian Origin.

PURPOSE: To test the retinal differentiation potential and to establish an optimized protocol for enriching retinal cells from an Indian origin, human embryonic stem cell (hESC) line, BJNhem20. METHODS: The BJNhem20 cells were cultured and expanded under feeder-free culture conditions. Differentiation was initiated by embryoid body (EB) formation and were cultured on Matrigel in neural induction medium (NIM) for 1 week and further maintained in retinal differentiation medium (RDM). After 1 month, the neuro-retinal progenitor clusters located at the center of pigmented retinal patches were picked and cultured as suspended neurospheres in RDM for 3 days and subsequently on Matrigel in neuro-retinal medium. The mildly pigmented, immature retinal pigmented epithelial (RPE) cells were picked separately and cultured on Matrigel in RPE medium (RPEM). After 1 week, the confluent neuro-retinal and RPE cultures were maintained in RDM for 2 to 3 months and characterized by immunofluorescence and RT-PCR. RESULTS: The BJNhem20 cells efficiently differentiated into both neuro-retinal and RPE cells. The early retinal progenitors expressed Nestin, GFAP, Pax6, Rx, MitfA, Chx10, and Otx2. Neuro-retinal cells expressed the neural markers, Map2, ß-III tubulin, acetylated tubulin and photoreceptor-specific markers, Crx, rhodopsin, recoverin, calbindin, PKC, NeuroD1, RLBP1, rhodopsin kinase, PDE6A, and PDE6C. Mature RPE cells developed intense pigmentation within 3 months and showed ZO-1 and Phalloidin staining at cell-cell junctions and expressed RPE65, tyrosinase, bestrophin1, Mertk, and displayed phagocytic activity. CONCLUSIONS: This study confirms the retinal differentiation potential of BJNhem20 cells and describes an optimized protocol to generate enriched populations of neuro-retinal and RPE cells.