Isolation of Reprogramming Intermediates During Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts

Christian M Nefzger; Sara Alaei; Jose M Polo

doi:10.1007/978-1-4939-2848-4_17

Isolation of Reprogramming Intermediates During Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts

Methods Mol Biol. 2015:1330:205-18. doi: 10.1007/978-1-4939-2848-4_17.

Authors

Christian M Nefzger¹, Sara Alaei¹, Jose M Polo²

Affiliations

¹ Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Level 3, STRIP 1, Building 75, Wellington Road, Clayton, VIC, 3800, Australia.
² Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Level 3, STRIP 1, Building 75, Wellington Road, Clayton, VIC, 3800, Australia. jose.polo@monash.edu.

PMID: 26621600
DOI: 10.1007/978-1-4939-2848-4_17

Abstract

Mature cells of the body can be reprogrammed towards a pluripotent state by forced expression of the transcription factors Oct-4, Klf-4, Sox2, and C-Myc (OKSM) at very low efficiency. To study the reprogramming process in detail the rare intermediates of the reaction need to be separated from the bulk population. Using a genetically engineered reprogrammable mouse strain we describe how to isolate intermediates from reprogramming cultures of mouse embryonic fibroblasts via antibody labeling of cell surface markers and fluorescence-activated cell sorting (FACS).

Keywords: Cell surface marker; Fluorescence-activated cell sorting; Induced pluripotent stem cells; Reprogrammable mouse model; Reprogramming intermediates.

MeSH terms

Animals
Cell Culture Techniques
Cell Differentiation
Cell Separation* / methods
Cellular Reprogramming*
Fibroblasts / cytology*
Flow Cytometry / methods
Induced Pluripotent Stem Cells / cytology*
Mice