Hill Matthew, Andrews-Pfannkoch Cynthia, Atherton Evan, Knudsen Travis, Trncic Emma, Marmorstein Alan D
Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, USA.
J Ocul Pharmacol Ther. 2024 Dec;40(10):680-687. doi: 10.1089/jop.2024.0130. Epub 2024 Oct 2.
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. 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. and were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs and were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of and in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, , and expression in iPSCs was generally uniform. The LOD for and in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔC found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. qPCR for and 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.
本研究的目的是开发一种用于诱导多能干细胞（iPSC）定向分化为视网膜色素上皮（RPE）细胞后iPSC残留检测的批量放行检测方法。对iPSC及其衍生的RPE进行RNA测序（RNA Seq），以鉴定在RPE细胞中下调的多能性标志物。然后应用定量实时PCR（qPCR）来评估iPSC衍生的RPE中的iPSC残留。通过将已知量的iPSC连续稀释到RPE悬浮液中进行加标试验来确定该检测方法的检测限（LOD）。[此处原文有缺失内容]和[此处原文有缺失内容]是RNA Seq鉴定出的在RPE中下调的8种多能性标志物中的两种。基于假基因和长链非编码RNA（lncRNA）的拷贝数和表达情况，选择[此处原文有缺失内容]和[此处原文有缺失内容]用于残留iPSC的qPCR检测。逆转录PCR表明，[此处原文有缺失内容]和[此处原文有缺失内容]在来自8个iPSC供体的21个克隆中表达普遍一致，而在来自5个供体系的RPE细胞中均无表达。基于qPCR，[此处原文有缺失内容]、[此处原文有缺失内容]和[此处原文有缺失内容]在iPSC中的表达通常是一致的。使用来自2个iPSC系的RPE加标试验确定了qPCR检测中[此处原文有缺失内容]和[此处原文有缺失内容]的LOD。对ΔΔC的分析发现，检测限<0.01%的细胞，相当于两个iPSC系中每10,000个RPE细胞中<1个iPSC。针对[此处原文有缺失内容]和[此处原文有缺失内容]的qPCR在iPSC衍生的RPE群体中检测到每10,000个残留iPSC中<1个，为批量放行检测提供了足够的iPSC残留LOD。
