Generation of germline-competent induced pluripotent stem cells

Area of Science:

• Stem cell biology
• Epigenetics
• Reproductive biology

Background:

• Induced pluripotent stem cells (iPS cells) offer a promising alternative to embryonic stem cells (ES cells).
• Previous methods using Fbx15 selection produced iPS cells with incomplete developmental potential and distinct epigenetic profiles.
• The role of Nanog in achieving full pluripotency and germline competence in iPS cells requires further investigation.

Purpose of the Study:

• To investigate whether selecting for Nanog expression in mouse fibroblasts-derived iPS cells enhances their pluripotency and germline competence.
• To compare the gene expression and DNA methylation patterns of Nanog-selected iPS cells with previously generated Fbx15-selected iPS cells.
• To assess the in vivo developmental potential and safety of Nanog-selected iPS cells, particularly concerning transgene silencing and tumor formation.

Main Methods:

• Retroviral transduction of mouse fibroblasts with Oct3/4, Sox2, c-Myc, and Klf4.
• Selection of iPS cells based on Nanog expression.
• Analysis of gene expression and DNA methylation patterns.
• Teratoma formation assays and chimera generation experiments.
• Assessment of transgene silencing and tumor development in offspring.

Main Results:

• Nanog-selected iPS cells exhibited increased similarity to ES cells in gene expression and DNA methylation patterns compared to Fbx15-selected iPS cells.
• The four reprogramming transgenes (Oct3/4, Sox2, c-Myc, Klf4) were significantly silenced in Nanog iPS cells.
• Seven out of ten Nanog iPS cell clones generated adult chimeras, with one clone demonstrating germline transmission.
• Tumor formation was observed in approximately 20% of offspring, linked to c-Myc transgene reactivation.

Conclusions:

• Selection for Nanog expression is a viable strategy to generate germline-competent iPS cells from mouse fibroblasts.
• Nanog-selected iPS cells demonstrate improved epigenetic and gene expression profiles resembling ES cells.
• While promising for regenerative medicine, the retroviral delivery of c-Myc necessitates caution due to potential oncogenic risks in clinical applications.