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Status |
Public on Feb 04, 2022 |
Title |
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 |
Organism |
Homo sapiens |
Experiment type |
Expression profiling by high throughput sequencing
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Summary |
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.
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Overall design |
Total RNA was isolated from self-renewing hESCs with low aSyn (Shef4, S9, S34) and high aSyn (S8, S37) expression, and from the same cell lines at Day 25 of cortical neuronal differentiation using the dual-Smad inhibition protocol
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Contributor(s) |
Natalwala A, Behbehani R, Kunath T |
Citation(s) |
35712660 |
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Submission date |
Feb 01, 2022 |
Last update date |
Jun 21, 2022 |
Contact name |
Tilo Kunath |
E-mail(s) |
tilo.kunath@ed.ac.uk
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Phone |
+44 (0) 131 651 9500
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Organization name |
University of Edinburgh
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Department |
Centre for Regenerative Medicine
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Street address |
5 LIttle France Drive
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City |
Edinburgh |
ZIP/Postal code |
EH16 4UU |
Country |
United Kingdom |
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Platforms (1) |
GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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Samples (17)
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Relations |
BioProject |
PRJNA802544 |