Improving single-cell cloning workflow for gene editing in human pluripotent stem cells - ScienceDirect

Stem Cell Research

Shondra M. Pruett-Miller^3,4²  Yi-Hsien Chen^1,2⁴ 

[1] Center for Advanced Genome Engineering, USA^4;Genome Engineering and iPSC Center, USA^2;St. Jude Children's Research Hospital, Department of Cell & Molecular Biology, Memphis 38105, USA^3;Washington University School of Medicine, Department of Genetics, St. Louis 63110, USA^1

关键词: Human pluripotent stem cells;    Embryonic stem cells;    Single-cell cloning;    Induced pluripotent stem cells;    hPSCs;    hESCs;    Genome engineering;    CRISPR-Cas9;   

DOI  :  10.1016/j.scr.2018.08.003

学科分类：生物科学（综合）

来源: Elsevier

【 摘 要 】

The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting. Stem-Flex and RevitaCell supplement, in combination with Geltrex or Vitronectin (VN), promote reliable single-cell growth in a feeder-free and defined environment. Characterization of final genome-edited clones reveals that pluripotency and normal karyotype are retained following this single-cell culture protocol. This time-efficient and simplified culture method paves the way for high-throughput hPSC culture and will be valuable for both basic research and clinical applications.

【 授权许可】

Unknown