Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.
Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.
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DOI：
10.1186/s13287-022-03141-2
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发表时间：
2022-09-05
影响因子：
7.5
通讯作者：
Sun, Xiaodong
中科院分区：
文献类型：
--
作者：
Zhang, Ting;Huang, Xianyu;Liu, Sujun;Bai, Xinyue;Zhu, Xinyue;Clegg, Dennis O.;Jiang, Mei;Sun, Xiaodong
人胚胎干细胞来源的视网膜色素上皮细胞（human embryonic stem cells derived retinal pigment epithelial cells，hESC-derived RPE）是一种很有前途的细胞替代疗法，可用于治疗视网膜变性疾病，但对RPE的冷冻保存研究有限。本研究旨在确定RPE冻存的最佳时相，以保持其解冻后的功能，并揭示RPE耐冷冻的机制。在接种后的不同时间点冷冻保存hESC衍生的RPE细胞。冻融后检测细胞的存活率、贴壁率、RPE标志基因表达、顶底极性、PEDF分泌、跨上皮阻力和吞噬能力。在3个时间点对RPE细胞进行RNA测序，鉴定差异表达基因，并利用基因本体论、京都基因和基因组百科全书、蛋白质-蛋白质相互作用分析等方法研究与RPE细胞抗冻性相关的关键通路或分子。在第2代第5天（P2 D5）冷冻的RPE在解冻后具有最高的细胞活力和附着。它们还保留了RPE标记基因的适当局部表达和生物学功能，如PEDF分泌、高跨上皮抗性和吞噬能力。RNA测序分析表明，RPE细胞在P2 D5表达高水平的细胞周期/DNA复制和ECM结合相关基因，以及THBS 1，这可能是一个可能的枢纽基因参与冷冻耐受性。我们还证实，在P2 D5的RPE细胞处于指数期，DNA复制活跃。我们建议在其指数期期间冷冻hESC衍生的RPE细胞在细胞活力和RPE细胞性质和功能的保存方面导致最佳的解冻后结果。细胞周期和ECM结合相关基因的高表达水平，特别是THBS 1，可能有助于该阶段更好的细胞恢复。在线版本包含补充材料，可通过10.1186/s13287-022-03141-2获得。
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance. hESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein–protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance. RPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication. We propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at this stage. The online version contains supplementary material available at 10.1186/s13287-022-03141-2.
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影响因子：
4.8
作者：
Bennis A;Jacobs JG;Catsburg LAE;Ten Brink JB;Koster C;Schlingemann RO;van Meurs J;Gorgels TGMF;Moerland PD;Heine VM;Bergen AA
通讯作者：
Bergen AA
影响因子：
7.5
作者：
Hongisto H;Ilmarinen T;Vattulainen M;Mikhailova A;Skottman H
通讯作者：
Skottman H
影响因子：
3.5
作者：
Brandl, Caroline;Zimmermann, Stephanie J.;Milenkovic, Vladimir M.;Rosendahl, Sibylle M. G.;Grassmann, Felix;Milenkovic, Andrea;Hehr, Ute;Federlin, Marianne;Wetzel, Christian H.;Helbig, Horst;Weber, Bernhard H. F.
通讯作者：
Weber, Bernhard H. F.
影响因子：
46.9
作者：
da Cruz, Lyndon;Fynes, Kate;Coffey, Peter J.
通讯作者：
Coffey, Peter J.
DOI：
10.1007/978-1-62703-080-9_20
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Mao, Yingyu;Finnemann, Silvia C
通讯作者：
Finnemann, Silvia C