Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and largescale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps ([ 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a Elena V. Grigor'eva and Tuyana B. Malankhanova contributed equally to this work.

During early development in female mammals, one of the two X chromosomes recruits a variety of protein complexes that establish repressive chromatin modifications and thus becomes transcriptionally silenced. This process is termed X chromosome inactivation (XCI). Imprinted XCI of the paternal X chromosome occurs in the extraembryonic lineages of some eutherian species (e.g., rodents). In the cells of the embryo proper, the choice of the X chromosome for XCI is random. In this study we compared the distribution of some histone modifications on metaphase spreads from extraembryonic endoderm and fibroblast cell lines in vole Microtus rossiaemeridionalis, which are examples of imprinted and random XCI, respectively. The X chromosome of M. rossiaemeridionalis bears a large constitutive heterochromatic block enriched with repeated DNA, making this species a useful model for studying chromatin structure. In vole fibroblasts and the majority of extraembryonic endoderm cells, the silencing of the inactive X chromosome appears to involve two types of facultative heterochromatin. The first is defined by H3K27 trimethylation and H2A ubiquitylation and colocalizes with previously described Xist RNA banding, whereas the second is associated with H3K9 trimethylation and the heterochromatic protein HP1. The block of constitutive heterochromatin on the M. rossiaemeridionalis X chromosome has the same pattern of chromatin modifications as the second type of facultative heterochromatin. The distribution of histone modifications, HP1 protein, and Xist RNA on vole inactive X chromosome is the same during both the imprinted and the random XCI.

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In rodent female mammals, there are two forms of X-inactivation – imprinted and random which take place in extraembryonic and embryonic tissues, respectively. The inactive X-chromosome during random X-inactivation was shown to contain two types of facultative heterochromatin that alternate and do not overlap. However, chromatin structure of the inactive X-chromosome during imprinted X-inactivation, especially at early stages, is still not well understood. In this work, we studied chromatin modifications associated with the inactive X-chromosome at different stages of imprinted X-inactivation in a rodent, Microtus levis. It has been found that imprinted X-inactivation in vole occurs in a species-specific manner in two steps. The inactive X-chromosome at early stages of imprinted X-inactivation is characterized by accumulation of H3K9me3, HP1, H4K20me3, and uH2A, resembling to some extent the pattern of repressive chromatin modifications of meiotic sex chromatin. Later, the inactive X-chromosome recruits trimethylated H3K27 and acquires the two types of heterochromatin associated with random X-inactivation.