SUMMARYIn the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to a terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of normal retina than was that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.
Summary
Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known if the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated iPSCs murine rod photoreceptors (r-iPSCs) and scored their ability to make retina using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs were more efficient at producing differentiated retina than embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had a reduction in amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod specific CTCF insulator protein binding sites may promote retinogenes in r-iPSCs. Taken together, our data suggest that the source of stem cells are important for producing retinal neurons in 3D organ cultures.
Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.
SUMMARYDiverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.
Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRSs) for schizophrenia (SCZ) and a clinical diagnosis of SCZ, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures in the patient-derived neurons that implicated altered Na+ channel function, action potential interspike interval, and gamma-aminobutyric acid–ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these SCZ patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.
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