Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing

Cells

Ralf Kühn¹  Anja Zimmer¹  Robin Friedrich²  Nilima Prakash²  Stefanie Brüggemann²  Parivash Nouri² 

[1] Genome Engineering & Disease Models, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany;Laboratory of Applied Genetics and Stem Cell Biology, Department Hamm 2, Hamm-Lippstadt University of Applied Sciences, 59063 Hamm, Germany;

关键词: iPSC;    NESTIN;    reporter;    CRISPR-Cas9;    neural stem cells;    midbrain dopamine neurons;   

DOI  :  10.3390/cells11020268

来源: DOAJ

【 摘 要 】

Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm.

【 授权许可】

Unknown