Long-term Follow-up of a Phase 1/2a Clinical Trial of a Stem Cell-Derived Bioengineered Retinal Pigment Epithelium Implant for Geographic Atrophy.

PURPOSE: To report long-term results from a phase 1/2a clinical trial assessment of a scaffold-based human embryonic stem cell-derived retinal pigmented epithelium (RPE) implant in patients with advanced geographic atrophy (GA). DESIGN: A single-arm, open-label phase 1/2a clinical trial approved by the United States Food and Drug Administration. PARTICIPANTS: Patients were 69-85 years of age at the time of enrollment and were legally blind in the treated eye (best-corrected visual acuity [BCVA], ≤ 20/200) as a result of GA involving the fovea. METHODS: The clinical trial enrolled 16 patients, 15 of whom underwent implantation successfully. The implant was administered to the worse-seeing eye with the use of a custom subretinal insertion device. The companion nonimplanted eye served as the control. The primary endpoint was at 1 year; thereafter, patients were followed up at least yearly. MAIN OUTCOME MEASURES: Safety was the primary endpoint of the study. The occurrence and frequency of adverse events (AEs) were determined by scheduled eye examinations, including measurement of BCVA and intraocular pressure and multimodal imaging. Serum antibody titers were collected to monitor systemic humoral immune responses to the implanted cells. RESULTS: At a median follow-up of 3 years, fundus photography revealed no migration of the implant. No unanticipated, severe, implant-related AEs occurred, and the most common anticipated severe AE (severe retinal hemorrhage) was eliminated in the second cohort (9 patients) through improved intraoperative hemostasis. Nonsevere, transient retinal hemorrhages were noted either during or after surgery in all patients as anticipated for a subretinal surgical procedure. Throughout the median 3-year follow-up, results show that implanted eyes were more likely to improve by > 5 letters of BCVA and were less likely to worsen by > 5 letters compared with nonimplanted eyes. CONCLUSIONS: This report details the long-term follow-up of patients with GA to receive a scaffold-based stem cell-derived bioengineered RPE implant. Results show that the implant, at a median 3-year follow-up, is safe and well tolerated in patients with advanced dry age-related macular degeneration. The safety profile, along with the early indication of efficacy, warrants further clinical evaluation of this novel approach for the treatment of GA. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

A haplotype-resolved genome assembly of the Nile rat facilitates exploration of the genetic basis of diabetes.

BACKGROUND: The Nile rat (Avicanthis niloticus) is an important animal model because of its robust diurnal rhythm, a cone-rich retina, and a propensity to develop diet-induced diabetes without chemical or genetic modifications. A closer similarity to humans in these aspects, compared to the widely used Mus musculus and Rattus norvegicus models, holds the promise of better translation of research findings to the clinic. RESULTS: We report a 2.5 Gb, chromosome-level reference genome assembly with fully resolved parental haplotypes, generated with the Vertebrate Genomes Project (VGP). The assembly is highly contiguous, with contig N50 of 11.1 Mb, scaffold N50 of 83 Mb, and 95.2% of the sequence assigned to chromosomes. We used a novel workflow to identify 3613 segmental duplications and quantify duplicated genes. Comparative analyses revealed unique genomic features of the Nile rat, including some that affect genes associated with type 2 diabetes and metabolic dysfunctions. We discuss 14 genes that are heterozygous in the Nile rat or highly diverged from the house mouse. CONCLUSIONS: Our findings reflect the exceptional level of genomic resolution present in this assembly, which will greatly expand the potential of the Nile rat as a model organism.

The effects of xeno-free cryopreservation on the contractile properties of human iPSC derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.

Vascular changes in diabetic retinopathy-a longitudinal study in the Nile rat.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies.

Subretinal Implantation of a Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium Monolayer in a Porcine Model.

The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 µm vs 292 ± 41 µm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.

Adipose Stem Cells Incorporated in Fibrin Clot Modulate Expression of Growth Factors.

PURPOSE: To evaluate the platelet capture rate of whole blood fibrin clots and the expression, secretion, and retention of the growth factors vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) from fibrin clots and to determine how these levels may be modulated by allogeneic adipose-derived stem cells (ASCs). METHODS: Whole blood from 10 human volunteers was transferred to a clotting device and the platelet capture rate determined. Two experimental conditions and 1 control were evaluated over 2 weeks in vitro. Clots made from human whole blood without ASCs, clot(-)ASC, were compared with clots with ASCs incorporated, clot(+)ASC, and a control group of synthetic polyethylene glycol gels with ASCs incorporated, control(+)ASCs. All conditions were examined for secretion and retention of VEGF, PDGF, and bFGF via enzyme-linked immunosorbent assay and immunohistochemistry. The analysis of platelet retention for clots made with this device was performed. RESULTS: Enzyme-linked immunosorbent assay analysis showed significantly higher (P < .001) secretion of VEGF in clot(+)ASC compared with clot(-)ASC or control(+)ASC. In contrast, clot(-)ASC produced soluble PDGF, and the addition of ASCs results in decreased soluble PDGF with concomitant increases in PDGF immunoreactivity of ASCs. Soluble bFGF levels were low in clot(-)ASC, and were found to increase at early time points in clot(+)ASC. Furthermore, bFGF immunoreactivity could be detected in clot(+)ASC, whereas no bFGF immunoreactivity is present in clot(-)ASC or control(+)ASC. Control(+)ASC displayed a spike in bFGF secretion at day 0, which may be due to a stress response elicited by the encapsulation process. Approximately 98% of available platelets in whole blood were concentrated in the clot on formation. CONCLUSIONS: Fibrin clots made by this method retain high concentrations of platelets, and when incorporated with ASCs show modulated secretion and immunoreactivity of VEGF, PDGF, and bFGF. CLINICAL RELEVANCE: Whole blood fibrin clots capture platelets and release growth factors, and the addition of ASCs increases VEGF release for up to 2 weeks after clot formation. This suggests that whole blood fibrin clots may be a viable scaffold and delivery vehicle for future stem cell treatments.

Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.

Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.

Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease.

Stem cells provide a potentially unlimited source of cells for treating a plethora of human diseases. Regenerative therapies for retinal degenerative diseases are at the forefront of translation to the clinic, with stem cell-derived retinal pigment epithelium (RPE)-based treatments for age-related macular degeneration (AMD) already showing promise in human patients. Despite our expanding knowledge of stem cell biology, methods for deriving cells, including RPE have remained inefficient. Thus, there has been a push in recent years to develop more directed approaches to deriving cells for therapy. In this concise review, we summarize recent efforts that have been successful in improving RPE derivation efficiency by directing differentiation from human pluripotent stem cells using developmental cues important for normal RPE specification and maturation in vivo. In addition, potential obstacles for clinical translation are discussed. Finally, we review how derivation of RPE from human induced pluripotent stem cells (hiPSCs) provides in vitro models for studying mechanisms of retinal disease and discovering new avenues for treatment.

Subretinal implantation of a monolayer of human embryonic stem cell-derived retinal pigment epithelium: a feasibility and safety study in Yucatán minipigs.

PURPOSE: A subretinal implant termed CPCB-RPE1 is currently being developed to surgically replace dystrophic RPE in patients with dry age-related macular degeneration (AMD) and severe vision loss. CPCB-RPE1 is composed of a terminally differentiated, polarized human embryonic stem cell-derived RPE (hESC-RPE) monolayer pre-grown on a biocompatible, mesh-supported submicron parylene C membrane. The objective of the present delivery study was to assess the feasibility and 1-month safety of CPCB-RPE1 implantation in Yucatán minipigs, whose eyes are similar to human eyes in size and gross retinal anatomy. METHODS: This was a prospective, partially blinded, randomized study in 14 normal-sighted female Yucatán minipigs (aged 2 months, weighing 24-35 kg). Surgeons were blinded to the randomization codes and postoperative and post-mortem assessments were performed in a blinded manner. Eleven minipigs received CPCB-RPE1 while three control minipigs underwent sham surgery that generated subretinal blebs. All animals except two sham controls received combined local (Ozurdex™ dexamethasone intravitreal implant) and systemic (tacrolimus) immunosuppression or local immunosuppression alone. Correct placement of the CPCB-RPE1 implant was assessed by in vivo optical coherence tomography and post-mortem histology. hESC-RPE cells were identified using immunohistochemistry staining for TRA-1-85 (a human marker) and RPE65 (an RPE marker). As the study results of primary interest were nonnumerical no statistical analysis or tests were conducted. RESULTS: CPCB-RPE1 implants were reliably placed, without implant breakage, in the subretinal space of the minipig eye using surgical techniques similar to those that would be used in humans. Histologically, hESC-RPE cells were found to survive as an intact monolayer for 1 month based on immunohistochemistry staining for TRA-1-85 and RPE65. CONCLUSIONS: Although inconclusive regarding the necessity or benefit of systemic or local immunosuppression, our study demonstrates the feasibility and safety of CPCB-RPE1 subretinal implantation in a comparable animal model and provides an encouraging starting point for human studies.

Initial Characterization of WDR5B Reveals a Role in the Proliferation of Retinal Pigment Epithelial Cells.

The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy.

Purpose: Isolating extracellular vesicles (EVs) with high yield, replicable purity, and characterization remains a bottleneck in the development of EV therapeutics. To address these challenges, the current study aims to establish the necessary framework for preclinical and clinical studies in the development of stem cell-derived intraocular EV therapeutics. Methods: Small EVs (sEVs) were separated from the conditioned cell culture medium (CCM) of the human embryogenic stem cell-derived fully polarized retinal pigment epithelium (hESC-RPE-sEV) by a commercially available microfluidic tangential flow filtration (TFF) device ExoDisc (ED) or differential ultracentrifugation (dUC). The scaling and concentration capabilities and purity of recovered sEVs were assessed. Size, number, and surface markers of sEVs were determined by orthogonal approaches using multiple devices. Results: ED yielded higher numbers of sEVs, ranging from three to eight times higher depending on the measurement device, compared to dUC using the same 5 mL of CCM input. Within the same setting, the purity of ED-recovered hESC-RPE-sEVs was higher than that for dUC-recovered sEVs. ED yielded a higher concentration of particles, which is strongly correlated with the input volume, up to 10 mL (r = 0.98, P = 0.016). Meanwhile, comprehensive characterization profiles of EV surface markers between ED- and dUC-recovered hESC-RPE-sEVs were compatible. Conclusions: Our study supports TFF as a valuable strategy for separating sEVs for the development of intraocular EV therapeutics. However, there is a growing need for diverse devices to optimize TFF for use in EV preparation. Using orthogonal approaches in EV characterization remains ideal for reliably characterizing heterogeneous EV.

Rescuing Photoreceptors in RPE Dysfunction-Driven Retinal Degeneration: The Role of Small Extracellular Vesicles Secreted from Retinal Pigment Epithelium.

Dysfunction of the retinal pigment epithelium (RPE) is a common shared pathology in major degenerative retinal diseases despite variations in the primary etiologies of each disease. Due to their demanding and indispensable functional roles throughout the lifetime, RPE cells are vulnerable to genetic predisposition, external stress, and aging processes. Building upon recent advancements in stem cell technology for differentiating healthy RPE cells and recognizing the significant roles of small extracellular vesicles (sEV) in cellular paracrine and autocrine actions, we investigated the hypothesis that the RPE-secreted sEV alone can restore essential RPE functions and rescue photoreceptors in RPE dysfunction-driven retinal degeneration. Our findings support the rationale for developing intravitreal treatment of sEV. We demonstrate that intravitreally delivered sEV effectively penetrate the full thickness of the retina. Xenogenic intraocular administration of human-derived EVs did not induce acute immune reactions in rodents. sEV derived from human embryonic stem cell (hESC)-derived fully differentiated RPE cells, but not sEV-depleted conditioned cell culture media (CCM minus sEV), rescued photoreceptors and their function in a Royal College of Surgeons (RCS) rat model. This model is characterized by photoreceptor death and retinal degeneration resulting from a mutation in the MerTK gene in RPE cells. From the bulk RNA sequencing study, we identified 447 differently expressed genes in the retina after hESC-RPE-sEV treatment compared with the untreated control. Furthermore, 394 out of 447 genes (88%) showed a reversal in expression toward the healthy state in Long-Evans (LE) rats after treatment compared to the diseased state. Particularly, detrimental alterations in gene expression in RCS rats, including essential RPE functions such as phototransduction, vitamin A metabolism, and lipid metabolism were partially reversed. Defective photoreceptor outer segment engulfment due to intrinsic MerTK mutation was partially ameliorated. These findings suggest that RPE-secreted sEV may play a functional role similar to that of RPE cells. Our study justifies further exploration to fully unlock future therapeutic interventions with sEV in a broad array of degenerative retinal diseases.

YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFß uptake in human pluripotent stem cell-derived cardiomyocytes.

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGFß. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM.

Mammalian Fuel Cells Produce Electric Current.

The increasing concern about climate change has led scientists around the world to develop clean energy technologies that may replace the traditional use of fossil fuels. A promising approach is the utilization of unicellular organisms as electron donors in bio-fuel cells. To date, this method has been limited to microorganisms such as bacteria, yeast, and microalgae. In this work, we show for the first time the concept of using mammalian cell cultures and organoids as electron donors in biofuel cells. We apply cyclic voltammetry to show that upon association of ARPE19 cells with the anode, they release reducing molecules to produce electricity. Furthermore, we apply 2D-fluorescence measurements and show that upon illumination, photosensitive stem cell-derived retinal organoids, which consist of rod photoreceptors and interneurons, secrete NADH and NADPH molecules that can donate electrons at the anode to produce photocurrent.

A Nile rat transcriptomic landscape across 22 organs by ultra-deep sequencing and comparative RNA-seq pipeline (CRSP).

RNA sequencing (RNA-seq) has been a widely used high-throughput method to characterize transcriptomic dynamics spatiotemporally. However, RNA-seq data analysis pipelines typically depend on either a sequenced genome and/or corresponding reference transcripts. This limitation is a challenge for species lacking sequenced genomes and corresponding reference transcripts. The Nile rat (Arvicanthis niloticus) has two key features - it is daytime active, and it is prone to diet-induced diabetes, which makes it more similar to humans than regular laboratory rodents. However, at the time of this study, neither a Nile rat genome nor a reference transcript set were available, making it technically challenging to perform large-scale RNA-seq based transcriptomic studies. This genome-independent work progressed concurrently with our generation of a Nile rat genome. A well-annotated genome requires several iterations of manually reviewing curated transcripts and takes years to achieve. Here, we developed a Comparative RNA-Seq Pipeline (CRSP), integrating a comparative species strategy independent of a specific sequenced genome or species-matched reference transcripts. We performed benchmarking to validate that our CRSP tool can accurately quantify gene expression levels. In this study, we generated the first ultra-deep (2.3 billion × 2 paired-end) Nile rat RNA-seq data from 59 biopsy samples representing 22 major organs, providing a unique resource and spatial gene expression reference for Nile rat researchers. Importantly, CRSP is not limited to the Nile rat species and can be applied to any species without prior genomic knowledge. To facilitate a general use of CRSP, we also characterized the number of RNA-seq reads required for accurate estimation via simulation studies. CRSP and documents are available at: https://github.com/pjiang1105/CRSP.

Transcriptomic clock predicts vascular changes of prodromal diabetic retinopathy.

Diabetic retinopathy is a common complication of long-term diabetes and that could lead to vision loss. Unfortunately, early diabetic retinopathy remains poorly understood. There is no effective way to prevent or treat early diabetic retinopathy until patients develop later stages of diabetic retinopathy. Elevated acellular capillary density is considered a reliable quantitative trait present in the early development of retinopathy. Hence, in this study, we interrogated whole retinal vascular transcriptomic changes via a Nile rat model to better understand the early pathogenesis of diabetic retinopathy. We uncovered the complexity of associations between acellular capillary density and the joint factors of blood glucose, diet, and sex, which was modeled through a Bayesian network. Using segmented regressions, we have identified different gene expression patterns and enriched Gene Ontology (GO) terms associated with acellular capillary density increasing. We developed a random forest regression model based on expression patterns of 14 genes to predict the acellular capillary density. Since acellular capillary density is a reliable quantitative trait in early diabetic retinopathy, and thus our model can be used as a transcriptomic clock to measure the severity of the progression of early retinopathy. We also identified NVP-TAE684, geldanamycin, and NVP-AUY922 as the top three potential drugs which can potentially attenuate the early DR. Although we need more in vivo studies in the future to support our re-purposed drugs, we have provided a data-driven approach to drug discovery.

Plasma metabolomics supports non-fasted sampling for metabolic profiling across a spectrum of glucose tolerance in the Nile rat model for type 2 diabetes.

Type 2 diabetes is a challenge in modern healthcare, and animal models are necessary to identify underlying mechanisms. The Nile rat (Arvicanthis niloticus) develops diet-induced diabetes rapidly on a conventional rodent chow diet without genetic or chemical manipulation. Unlike common laboratory models, the outbred Nile rat model is diurnal and has a wide range of overt diabetes onset and diabetes progression patterns in both sexes, better mimicking the heterogeneous diabetic phenotype in humans. While fasted blood glucose has historically been used to monitor diabetic progression, postprandial blood glucose is more sensitive to the initial stages of diabetes. However, there is a long-held assumption that ad libitum feeding in rodent models leads to increased variance, thus masking diabetes-related metabolic changes in the plasma. Here we compared repeatability within triplicates of non-fasted or fasted plasma samples and assessed metabolic changes relevant to glucose tolerance in fasted and non-fasted plasma of 8-10-week-old male Nile rats. We used liquid chromatography-mass spectrometry lipidomics and polar metabolomics to measure relative metabolite abundances in the plasma samples. We found that, compared to fasted metabolites, non-fasted plasma metabolites are not only more strongly associated with glucose tolerance on the basis of unsupervised clustering and elastic net regression model, but also have a lower replicate variance. Between the two sampling groups, we detected 66 non-fasted metabolites and 32 fasted metabolites that were associated with glucose tolerance using a combined approach with multivariable elastic net and individual metabolite linear models. Further, to test if metabolite replicate variance is affected by age and sex, we measured non-fasted replicate variance in a cohort of mature 30-week-old male and female Nile rats. Our results support using non-fasted plasma metabolomics to study glucose tolerance in Nile rats across the progression of diabetes.

Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

Efficient derivation of transgene-free porcine induced pluripotent stem cells enables in vitro modeling of species-specific developmental timing.

Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ∼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.