Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse

Area of Science:

• Stem cell biology
• Molecular biology
• Cellular reprogramming

Background:

• Induced pluripotent stem (iPS) cells are generated by ectopic expression of specific transcription factors (Oct4, Sox2, c-Myc, Klf4) in somatic cells like fibroblasts.
• The precise molecular events and temporal sequence during nuclear reprogramming remain incompletely understood.

Purpose of the Study:

• To elucidate the molecular events and identify key markers during the process of nuclear reprogramming leading to iPS cell generation.
• To define intermediate cell populations during the transition to a self-sustaining pluripotent state.

Main Methods:

• Utilized doxycycline-inducible vectors to control the expression of reprogramming factors.
• Monitored molecular marker expression (Thy1, SSEA-1, endogenous Oct4, Sox2, telomerase, X chromosome reactivation) over time.
• Employed cell sorting based on identified markers to enrich for cells with iPS cell potential.

Main Results:

• Exogenous factor expression is necessary for approximately 10 days before cells achieve self-sustaining pluripotency.
• Identified distinct early (Thy1 downregulation, SSEA-1 upregulation) and late (endogenous Oct4/Sox2 reactivation, telomerase activation, X chromosome reactivation) molecular events.
• Marker-based cell sorting significantly enriched populations capable of becoming iPS cells.

Conclusions:

• Factor-induced reprogramming is a progressive process characterized by defined intermediate cell stages.
• Intermediate cell populations identified by specific markers are enriched for cells poised to achieve pluripotency.
• Understanding these sequential events and markers aids in optimizing and analyzing the iPS cell generation process.