Heine, V. | bioRxiv Science

bioRxiv2019

Quantitative proteomic alterations of human iPSC-based neuronal development indicate early onset of Rett syndrome.

Rett syndrome (RTT) is a progressive neurodevelopmental disease often caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). The mechanisms by which impaired MeCP2 induces the pathological abnormalities in the brain are not understood. To understand the molecular mechanisms involved in disease, we used an RTT patient induced pluripotent stem cell (iPSC)-based model and applied an in-depth high-resolution quantitative mass spectrometry-based analysis during early stages of neuronal development. Our data provide evidence of proteomic alteration at developmental stages long before the phase that symptoms of RTT syndrome become apparent. Differences in expression profiles became more pronounced from early to late neural stem cell phases, although proteins involved in immunity, metabolic processes and calcium signaling were already affected at initial stages. These results can help development of new biomarkers and therapeutic approaches by selectively target the affected proteins in RTT syndrome.

neuroscience↗

bioRxiv2019

Human and mouse iPSC-derived astrocyte subtypes reveal vulnerability in Vanishing White Matter

Astrocytes gained attention as important players in neurological disease, including a number of leukodystrophies. Several studies explored the generation of induced pluripotent stem cell-derived astrocytes for drug screening and regenerative studies. Developing robust models of patient induced pluripotent stem cells is challenged by high variability due to diverse genetic backgrounds and long-term culture procedures. While human models are of special interest, mouse-based models have the advantage that for them these issues are less pronounced. Here we present astrocyte differentiation protocols for both human and mouse induced pluripotent stem cells to specifically induce grey and white matter astrocytes. Both subtypes expressed astrocyte-associated markers, had typical astrocyte morphologies, and gave a reactive response to stress. Importantly, the grey and white matter-like astrocytes differed in size, complexity of processes, and expression profile, conform primary grey and white matter astrocytes. The newly presented mouse and human stem cell-based models for the leukodystrophy Vanishing White Matter replicated earlier findings, such as increased proliferation, decreased OPC maturation and modulation by hyaluronidase. We studied intrinsic astrocyte subtype vulnerability in Vanishing White Matter in both human and mouse cells. Oligodendrocyte maturation was specifically inhibited in cultures with Vanishing White Matter white matter-like astrocytes. By performing RNA sequencing, we found more differentially regulated genes in the white than in the grey matter-like astrocytes. Human and mouse astrocytes showed the same affected pathways, although human white matter-like astrocytes presented human-specific disease mechanisms involved in Vanishing White Matter. Using both human and mouse induced pluripotent stem cells, our study presents protocols to generate white and grey matter-like astrocytes, and shows astrocyte subtype-specific defects in Vanishing White Matter. While mouse induced pluripotent stem cell-based cultures may be less suitable to mimic human astrocyte subtype- or patient-specific changes, they might more robustly represent disease mutation-related cellular phenotypes as the cells are derived from inbred mice and the protocols are faster. The presented models give new tools to generate astrocyte subtypes for in vitro disease modeling and in vivo regenerative applications.

neuroscience↗