患者来源 iPS 细胞的视网膜分化倾向:自体细胞替代的意义。

Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement.

机构信息

Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

出版信息

Stem Cells Transl Med. 2023 Jun 15;12(6):365-378. doi: 10.1093/stcltm/szad028.

Abstract

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.

摘要

在使用之前,新生成的诱导多能干细胞(iPSC)应进行彻底验证。虽然存在旨在评估效力、遗传完整性和无菌性的出色验证和放行测试检测方法,但它们没有能力预测细胞类型特异性分化能力。选择具有有限产生高质量可移植细胞能力的 iPSC 系,会对宝贵的临床制造资源造成巨大压力。本研究的目的是确定 cGMP 来源的患者 iPSC 系之间视网膜分化能力的变异性程度和根本原因。反过来,我们的目标是开发一种放行测试检测方法,可用于增强广泛使用的 ScoreCard 试剂盒。从 15 名患者(14-76 岁)中生成 iPSC,分化为视网膜类器官,并根据其视网膜分化能力进行评分。尽管视网膜分化倾向存在显著差异,但 RNA 测序显示分化前患者来源的 iPSC 系之间存在显著相似性。在分化的第 7 天,可以检测到基因表达的显著差异。Ingenuity 通路分析显示与多能性和早期细胞命运决定相关的途径受到干扰。例如,好的和差的生产者的 OCT4 和 SOX2 效应基因表达明显不同。通过 RNA 测序鉴定的基因的 QPCR 检测方法以掩蔽方式开发并使用 8 名独立患者的 iPSC 进行验证。一组 14 个基因,包括视网膜细胞命运标记物 RAX、LHX2、VSX2 和 SIX6(在好的生产者中均升高),被发现可预测视网膜分化倾向。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ed9/10267581/0bd455121c9d/szad028_fig5.jpg

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索