Retinal Cell Type DNA Methylation and Histone Modifications Predict Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures.

Diverse 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.

The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis.

In 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 terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of the normal retina than that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

Spinal muscular atrophy astrocytes exhibit abnormal calcium regulation and reduced growth factor production.

Spinal muscular atrophy (SMA) is a genetic disorder caused by the deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Although motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA-induced pluripotent stem cells exhibit morphological and cellular changes indicative of activation before overt motor neuron loss. Furthermore, our in vitro studies show mis-regulation of basal calcium and decreased response to adenosine triphosphate stimulation indicating abnormal astrocyte function. Together, for the first time, these data show early disruptions in astrocytes that may contribute to SMA disease pathology.

Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain.

Hindlimb pain models developed in rats have been transposed to mice, but assumed sciatic nerve neuroanatomic similarities have not been examined. We compared sciatic nerve structural organization in mouse strains (C57BL/6J, DBA/2J, and B6129PF2/J) and rat strains (Wistar, Brown Norway, and Sprague-Dawley). Dissection and retrograde labeling showed mouse sciatic nerve origins predominantly from the third lumbar (L3) and L4 spinal nerves, unlike the L4 and L5 in rats. Proportionate contributions by each level differed significantly between strains in both mice and rats. Whereas all rats had six lumbar vertebrae, variable patterns in mice included mostly five vertebrae in DBA/2J, mostly six vertebrae in C57BL/6J, and a mix in B6129PF2/J. Mice with a short lumbar vertebral column showed a rostral shift in relative contributions to the sciatic nerve by L3 and L4. Ligation of the mouse L4 nerve created hyperalgesia similar to that in rats after L5 ligation, and motor changes were similar after mouse L4 and rat L5 ligation (foot cupping) and after mouse L3 and rat L4 ligation (flexion weakness). Thus, mouse L3 and L4 neural segments are anatomically and functionally homologous with rat L4 and L5 segments. Neuronal changes after distal injury or inflammation should be sought in the mouse L3 and L4 ganglia, and the spinal nerve ligation model in mice should involve ligation of the L4 nerve while L3 remains intact. Strain-dependent variability in segmental contributions to the sciatic nerve may account in part for genetic differences in pain behavior after spinal nerve ligation.