Protocol for generating transgene-free naive human induced pluripotent stem cells from somatic cells using modified...

Area of Science:

• Stem Cell Biology
• Molecular Biology
• Virology

Background:

• Sendai virus (SeV) vectors are effective for generating induced pluripotent stem cells (iPSCs).
• Existing protocols may require optimization for generating naive human iPSCs from various somatic cell types.
• Transgene-free reprogramming is crucial for safe iPSC applications.

Purpose of the Study:

• To present a detailed protocol for generating transgene-free naive human iPSCs.
• To utilize a modified SeV vector system for reprogramming human dermal fibroblasts (HDFs) and human peripheral mononuclear cells (PBMCs).
• To establish a reliable method for generating and maintaining naive iPSCs.

Main Methods:

• Thawing and reseeding HDFs or PBMCs.
• Infection with a modified SeV vector.
• Culture on irradiated mouse embryonic fibroblasts (iMEFs) in t2iLGö+Y medium.
• Passaging, SeV vector removal, and cryopreservation of naive iPSCs.

Main Results:

• Successful generation of transgene-free naive human iPSCs from HDFs and PBMCs.
• Demonstration of a complete protocol from cell thawing to cryopreservation.
• The modified SeV vector system facilitates efficient reprogramming.

Conclusions:

• The presented protocol offers a robust method for generating transgene-free naive human iPSCs.
• This approach is applicable to primary somatic cells like HDFs and PBMCs.
• The protocol supports the generation and maintenance of high-quality naive iPSCs for research and therapeutic applications.