Characterization and staging of outer plexiform layer development in human retina and retinal organoids.

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (P<0.0001). Overall, this study defines stages of human OPL development through mid-gestation and establishes HROs as a model system that recapitulates key aspects of human photoreceptor-bipolar cell synaptogenesis in vitro.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

Aberrant gene expression yet undiminished retinal ganglion cell genesis in iPSC-derived models of optic nerve hypoplasia.

BACKGROUND: Optic nerve hypoplasia (ONH), the leading congenital cause of permanent blindness, is characterized by a retinal ganglion cell (RGC) deficit at birth. Multifactorial developmental events are hypothesized to underlie ONH and its frequently associated neurologic and endocrine abnormalities; however, environmental influences are unclear and genetic underpinnings are unexplored. This work investigates the genetic contribution to ONH RGC production and gene expression using patient induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs). MATERIALS AND METHODS: iPSCs produced from ONH patients and controls were differentiated to ROs. RGC genesis was assessed using immunofluorescence and flow cytometry. Flow-sorted BRN3+ cells were collected for RNA extraction for RNA-Sequencing. Differential gene expression was assessed using DESeq2 and edgeR. PANTHER was employed to identify statistically over-represented ontologies among the differentially expressed genes (DEGs). DEGs of high interest to ONH were distinguished by assessing function, mutational constraint, and prior identification in ONH, autism and neurodevelopmental disorder (NDD) studies. RESULTS: RGC genesis and survival were similar in ONH and control ROs. Differential expression of 70 genes was identified in both DESeq2 and edgeR analyses, representing a ~ 4-fold higher percentage of DEGs than in randomized study participants. DEGs showed trends towards over-representation of validated NDD genes and ONH exome variant genes. Among the DEGs, RAPGEF4 and DMD had the greatest number of disease-relevant features. CONCLUSIONS: ONH genetic background was not associated with impaired RGC genesis but was associated with DEGs exhibiting disease contribution potential. This constitutes some of the first evidence of a genetic contribution to ONH.

Methotrexate and aminopterin exhibit similar in vitro and in vivo preclinical activity against acute lymphoblastic leukaemia and lymphoma.

Due to the development of neurological toxicity and resistance to methotrexate (MTX), other antifolates have been evaluated for its potential replacement in the treatment of childhood acute lymphoblastic leukaemia (ALL). Aminopterin (AMT) has been suggested to provide clinical advantages over MTX and other antifolates. AMT activity, compared with MTX, was evaluated in ALL and lymphoma preclinical models. The minimum survival fraction at the range of concentrations tested was lower with AMT than with MTX in 3 out of 15 cell lines. Both AMT and MTX significantly extended the event-free survival of mice bearing 3 out of 4 xenografts with equivalent activity.

Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.

Purpose: Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Methods: Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue. Results: We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae. Conclusions: Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.