Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement.
Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement.
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DOI：
10.1093/stcltm/szad028
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发表时间：
2023-06-15
影响因子：
6
通讯作者：
中科院分区：
文献类型：
--
作者：
在使用之前，新产生的诱导多能干细胞（iPSC）应经过彻底验证。虽然存在设计用于评价效价、遗传完整性和无菌性的优良验证和放行检测测定，但它们不具有预测细胞类型特异性分化能力的能力。选择生产高质量可移植细胞的能力有限的iPSC系对宝贵的临床制造资源造成了重大压力。本研究的目的是确定cGMP衍生的患者iPSC系之间视网膜分化能力的变异性的程度和根本原因。反过来，我们的目标是开发一种可用于增强广泛使用的ScoreCard面板的放行检测方法。从15名患者（14-76岁）产生IPSC，分化成视网膜类器官，并基于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测序揭示了分化前患者来源的iPSC系之间的显著相似性。在分化7天时，可以检测到基因表达的显著差异。免疫途径分析揭示了与多能性和早期细胞命运定型相关的途径的扰动。例如，好的和差的生产者具有明显不同的OCT 4和SOX 2效应基因表达。开发了靶向通过RNA测序鉴定的基因的QPCR测定，并使用来自8名独立患者的iPSC以掩蔽方式进行验证。发现包括视网膜细胞命运标志物RAX、LHX 2、VSX 2和SIX 6（在良好生产者中均升高）的14个基因的子集预测视网膜分化倾向。
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.
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影响因子：
14.9
作者：
Frankish A;Diekhans M;Ferreira AM;Johnson R;Jungreis I;Loveland J;Mudge JM;Sisu C;Wright J;Armstrong J;Barnes I;Berry A;Bignell A;Carbonell Sala S;Chrast J;Cunningham F;Di Domenico T;Donaldson S;Fiddes IT;García Girón C;Gonzalez JM;Grego T;Hardy M;Hourlier T;Hunt T;Izuogu OG;Lagarde J;Martin FJ;Martínez L;Mohanan S;Muir P;Navarro FCP;Parker A;Pei B;Pozo F;Ruffier M;Schmitt BM;Stapleton E;Suner MM;Sycheva I;Uszczynska-Ratajczak B;Xu J;Yates A;Zerbino D;Zhang Y;Aken B;Choudhary JS;Gerstein M;Guigó R;Hubbard TJP;Kellis M;Paten B;Reymond A;Tress ML;Flicek P
通讯作者：
Flicek P
DOI：
10.1007/978-1-0716-2010-6_16
发表时间：
2022-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Hau, Kwan-Leong;Lane, Amelia;Cheetham, Michael E
通讯作者：
Cheetham, Michael E
影响因子：
64.8
作者：
Kim, Jeong Beom;Greber, Boris;Schoeler, Hans R.
通讯作者：
Schoeler, Hans R.
影响因子：
46.9
作者：
da Cruz, Lyndon;Fynes, Kate;Coffey, Peter J.
通讯作者：
Coffey, Peter J.
影响因子：
4.8
作者：
Baghbaderani, Behnam Ahmadian;Syama, Adhikarla;Rao, Mahendra S.
通讯作者：
Rao, Mahendra S.