Optimized surface markers for the prospective isolation of high-quality hiPSCs using flow cytometry selection

Area of Science:

• Stem Cell Biology
• Cellular Reprogramming
• Biotechnology

Background:

• Human induced pluripotent stem cell (hiPSC) derivation and selection are inefficient.
• Current methods require extensive characterization, limiting high-throughput (HTP) approaches.
• Previous work enhanced hiPSC survival and HTP derivation using small molecules and flow cytometry.

Purpose of the Study:

• To develop an enhanced protocol for efficient and automated isolation of bona fide hiPSCs.
• To optimize cell surface marker combinations for fluorescence-activated cell sorting (FACS)-based selection.
• To identify pivotal markers for hiPSC identification during reprogramming.

Main Methods:

• Utilized a cocktail of small molecules to improve hiPSC survival and stability in single-cell culture.
• Employed flow cytometry cell sorting for HTP-derivation of hiPSCs.
• Optimized a combination of cell surface markers, including CD30, SSEA4, and TRA-1-81, for FACS selection.

Main Results:

• Depletion of CD30-positive cells nearly abolished NANOG and OCT4 positive subpopulations, indicating CD30 as a key marker for pluripotency.
• Combining CD30 with SSEA4 and TRA-1-81 in FACS significantly enhanced the specificity and efficiency of hiPSC selection.
• The developed method enables efficient, automated, and prospective isolation of high-quality hiPSCs.

Conclusions:

• The optimized FACS-based selection protocol using CD30, SSEA4, and TRA-1-81 significantly improves hiPSC isolation efficiency and specificity.
• This method facilitates automated, high-throughput derivation of bona fide hiPSCs.
• The findings provide a robust tool for advancing stem cell research and regenerative medicine applications.