ArticleFrontiers in cell and developmental biology2019
An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells.
Article in Frontiers in cell and developmental biology, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Article
- Single-cell cloning and its approaches.Frontiers in cell and developmental biology · 2025Review
- Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy.BMC biotechnology · 2024Article
- Article
- Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling.Stem cell reports · 2023Article
- Understanding blood development and leukemia using sequencing-based technologies and human cell systems.Frontiers in molecular biosciences · 2023Review
- A Fast and Reliable Method to Generate Pure, Single Cell-derived Clones of Mammalian Cells.Bio-protocol · 2022Article
- Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.Small (Weinheim an der Bergstrasse, Germany) · 2022Article
- Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells.Frontiers in bioengineering and biotechnology · 2022Article
- Article
- A CRISPR/Cas9-based method for targeted DNA methylation enables cancer initiation in B lymphocytes.Advanced genetics (Hoboken, N.J.) · 2021Article
- Single Cell Metabolomics: A Future Tool to Unmask Cellular Heterogeneity and Virus-Host Interaction in Context of Emerging Viral Diseases.Frontiers in microbiology · 2020Review
Corrections and comments
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Authors and funding
1 author.
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Abstract
Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7-10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1-2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.
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