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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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