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Transcriptomic analysis of undifferentiated human embryonic stem cells (hESCs) and day-25 differentiated cortical neuronal progenitor cells from 5 isogenic hESC lines with low and high levels of alpha-synuclein expression

ABSTRACT: Purpose: To compare the cortical neuroonal differentiation capacity of clonal isogenic hESC lines with different levels of alpha-synuclein (aSyn) expression. Methods 1: Shef4 hESCs was used as a parental line to create an allelic series of clonal transgenic hESC lines expressing a human SNCA (encoding aSyn) contruct. Clonal transgenic lines with high (S8, S37) and low (S9, S34) aSyn expression were established and characterized. Methods 2: hESCs were cultured on Laminin-521 (BioLamina) in StemMACS iPS-Brew XF self-renewal media (Miltenyi) prior to lifting for isolation of total RNA. Methods 3: Cortical neuronal progenitor cells were differentiated from hESCs on Laminin-111 coated (Biolamina) 24-well plates at an initial plating density of 80,000 cells/cm2. Differentiation commenced in neural induction media (NIM) consisting of 50% DMEM/F12 (ThermoFisher Scientific), 50% Neurobasal Media (ThermoFisher Scientific), B27 supplement with Retinoic Acid (ThermoFisher Scientific), N2 supplement (ThermoFisher Scientific) and 2 mM L-Glutamine (ThermoFisher Scientific), 10μM SB431542 (Tocris) and 100 nM LDN-193189 (Miltenyl Biotec). From day 4 onwards, the base media was changed to 50% NIM, 25% DMEM/F12 and 25% Neurobasal Media. SB431542 (10 μM) and LDN-193189 (100 nM) were present for the first 12 days of differentiation. Cells were lifted and re-plated at day 12 and day 17 with Collagenase Type IV (Life Technologies). At day 25, differentiated cells were lifted for total RNA isolation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE195877 | GEO | 2022/02/04

REPOSITORIES: GEO

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Project description:Studies of human embryonic stem cells (hESCs) commonly describe the non-functional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of microRNAs, including hESC-specific microRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC microRNA cluster. Most importantly, we show that the hESC-enriched microRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and thus demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of microRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. Karyotypically normal hESC lines CCTL-14 (46, XX, at passages 20M-bM-^@M-^S30 and 242M-bM-^@M-^S250, respectively) was used in this study (for detailed characterization of hESC lines see International Stem Cell Registry: http://www.iscr-admin). For UVC irradiation, culture media were aspirated and cells were washed once with phosphate-buffered saline (PBS) without Mg2+ and Ca2+ (pH 7.4). For each time point, nine culture dishes were independently irradiated with a dose of 3 J/m2. Cells were then further incubated in culture media (37M-BM-0C/5% CO2) until they were harvested at 4, 8, and 16 hrs, respectively, post-UVC treatment. Differentiation and culture of NPCs derived from hESCs To induce neural differentiation, we utilized a protocol based on embryoid body (EB) formation with simultaneous all-trans-retinoic acid (RA) treatment. Colonies of hESCs were transferred to a non-adherent cell culture dish in the presence of differentiation media [DMEM/F12:Neurobasal 1:1, N2, B27 (Invitrogen), 2 mmol/l L-Glutamine (all media components from Invitrogen, Carlsbad, CA, USA), 1M-CM-^W MEM non-essential amino acids, 0.5% penicillin-streptomycin, 100 M-BM-5mol/l M-NM-2-2 mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml FGF-2 (PeproTech, Rocky Hill, NJ, USA)]. RA was added (Sigma-Aldrich, concentration 0.5 M-BM-5mol/l) into fresh differentiation media on days 6, 10, and 14 after the beginning of EB development. On day 16, cells were dissociated using Accutase (Sigma-Aldrich) and plated on a gelatin-coated cell culture dish in maintenance media (DMEM/F12, N2, B27, L-Glutamine, MEM non-essential amino acids, 0.5% penicillin-streptomycin, 100 M-BM-5mol/l M-NM-2-2 mercaptoethanol, and 20 ng/ml FGF-2). Cells were passaged twice and subsequently harvested for RNA isolation.

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Transcriptomic analysis of undifferentiated human embryonic stem cells (hESCs) and day-25 differentiated cortical neuronal progenitor cells from 5 isogenic hESC lines with low and high levels of alpha-synuclein expression

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