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| Status |
Public on Oct 03, 2024 |
| Title |
Detection Of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
PURPOSE: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. METHODS: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. RESULTS: ZSCAN10 and Lin28a were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and Lin28a were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and Lin28a in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10 and Lin28a expression in iPSCs was generally uniform. The LOD for ZSCAN10 and Lin28a in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of DDCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC / 10,000 RPE cells in both iPSC lines. CONCLUSIONS: qPCR for ZSCAN10 and Lin28a detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.
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| Overall design |
RNA Seq and differential gene expression analysis was performed by Azenta Life Sciences (Chelmsford, Massachusetts) on 3 samples each of 22/1 iPSCs and 3 samples of RPE cells derived from them. Reads were aligned and mapped to the HG38 genome assembly.
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| Contributor(s) |
Hill M, Andrews-Pfannkoch C, Atherton E, Knudsen T, Trncic E, Marmorstein AD |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Sep 04, 2024 |
| Last update date |
Oct 03, 2024 |
| Contact name |
Alexandra Krivonos |
| Organization name |
Mayo Clinic
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| Department |
Quantitative Health Sciences
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| Street address |
200 1st St SW
|
| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55905 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA1156779 |
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