Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry

作     者：Dmitry A Ovchinnikov Jennifer P Turner Drew M Titmarsh Nilay Y Thakar Dong Choon Sin Justin J Cooper-White Ernst J Wolvetang 

作者机构：Stem Cell Engineering GroupAustralian Institute for Bioengineering and NanotechnologyUniversity of QueenslandBrisbane 4072QueenslandAustralia Tissue Engineering and Microfluidics LaboratoryAustralian Institute for Bioengineering and NanotechnologyUniversity of QueenslandBrisbane 4072QueenslandAustralia School of Chemical EngineeringUniversity of Queensland Brisbane 4072QueenslandAustralia 

出 版 物：《World Journal of Stem Cells》 (世界干细胞杂志（英文版）（电子版）)

年 卷 期：2012年第4卷第7期

页      面：71-79页

学科分类：1001[医学-基础医学(可授医学、理学学位)] 100101[医学-人体解剖与组织胚胎学] 10[医学] 

基　　金：the Australian Stem CellCentre (ASCC), UQ AIBN Challenge Grant Scheme for financial support the ASCC’s StemCore pluripotent stem cell core facility for the provision of hES cell lines and logistical support 

主　　题：Human embryonic stem cells Fluorescent marker mCherry Pluripotency Cellular motility 

摘      要：AIM:The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS:Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays andembryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy. RESULTS:Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripo-tency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to singlecell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival. CONCLUSION:The MEL2-mCherry hESC line c