Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Overview

This protocol describes the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Key Study Components

Area of Science

• Stem Cell Biology
• Genetic Engineering
• Cell Culture Techniques

Background

• Induced pluripotent stem cells (iPSCs) are valuable for research and therapy.
• Footprint-free iPSCs avoid integration issues associated with traditional methods.
• CRISPR/Cas9 technology allows precise genome editing.
• Characterization of modified clones is essential for validating genetic changes.

Purpose of Study

• To generate footprint-free iPSCs from human pancreatic cells.
• To utilize CRISPR/Cas9 for editing these cells.
• To characterize the resulting modified single-cell clones.

Methods Used

• Coating six-well plates with cold collagen.
• Plating early passage human primary pancreatic cells in Prigrow III medium.
• Transduction with Sendai vector tubes for genome editing.
• Characterization of edited iPSC clones for reliability and mosaicism.

Main Results

• Successful generation of footprint-free iPSCs.
• High reliability in generating clonal lines of edited iPSCs.
• No evidence of mosaicism in the modified clones.
• Validated the effectiveness of CRISPR/Cas9 ribonucleoproteins.

Conclusions

• This method provides a reliable approach for generating and editing iPSCs.
• Footprint-free iPSCs can be used for various applications in research.
• CRISPR/Cas9 ribonucleoproteins are effective for precise genome editing.

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