Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons, both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes which are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor, p21, in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor to the pathology of Parkinson's disease. hESC shows normal appearance. Scale bar: 200 µm. (D) Immunolabeling of DA precursor markers in WT and SATB1 KO hESC derived DA neurons after 16 days of differentiation reveals no difference in marker expression. (E) Depiction of spontaneous action potentials of WT and SATB1 KO DA neurons at different time points show that both genotypes differentiate into mature DA neurons between day 35 and 40. Scale bar: 20 mV, 2 s. (F) DA neurons at day 40 show significant differences in maintenance of response to positive current injections. Scale bar: 20 mV, 200 ms. (G) Longitudinal comparison of cell survival of WT vs. SATB1 KO DA neurons revealed a significant reduction in SATB1 KO survival between day 30 and 40, reaching a plateau at ~50%. Data are represented as mean ± SEM. (H) Quantification of neurite morphology and complexity in WT and SATB1 KO DA neurons. Data are represented as mean ± SEM. (I) Triple immunolabeling of cortical shows that both WT as well as SATB1 KO neurons express the essential markers for cortical neurons. Scale bar: 50 µm. (J) SATB1 KO cortical neurons show no significant change in survival during differentiation. Data are represented as mean ± SEM. (K) Quantification of neurite morphology and complexity in WT and SATB1 KO CTX neurons. (L)Representative western blot and quantification depicts that WT DA neurons express significantly higher levels of SATB1 than WT cortical neurons, suggesting a higher demand in DA neurons. Data are represented as mean ± SEM. See also Figure S1.

Running Title: iPSC-based restoration of ovarian function following gonadotoxic chemotherapy iPSC -based restoration of ovarian function following gonadotoxic chemotherapy 2 Abstract Many oncologic therapies given to young women are gonadotoxic and associated with diminished ovarian reserve, increased risk of permanent sterility, and premature menopause. Previously, we reported the derivation of steroidogenic ovarian cells from induced pluripotent and embryonic stem cells. Derived cells not only produced reproductive hormones, but also displayed markers of ovarian tissue and primordial gametes. Here, we describe that human follicular fluid, when added to our stem cell differentiation system, enhances the steroidogenic potential of derived cells and increases the subpopulation of cells that differentiate to express the ovarian and germ cell markers GJA1 and ZP1, respectively. More importantly, using an in vivo model of chemotherapy-induced premature ovarian insufficiency in subfertile nude mice, we demonstrate that orthotopic implantation of these derived cells restores ovarian hormonal production and produces functional stem cell-derived germ cells. Collectively, these data support the hypothesis that stem cell-derived steroidogenic ovarian tissue could be used to promote neo-gametogenesis and treat premature menopause.iPSC -based restoration of ovarian function following gonadotoxic chemotherapy

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