Reprogramming Roadblocks Are System Dependent.
Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog(-/-) fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.
Eleni
E
Chantzoura
Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland.
Stavroula
S
Skylaki
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.
Sergio
S
Menendez
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Shin-Il
SI
Kim
Laboratory for Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 1, 171 77 Stockholm, Sweden.
Anna
A
Johnsson
Laboratory for Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 1, 171 77 Stockholm, Sweden.
Sten
S
Linnarsson
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Hakubi Center for Advanced Research, Kyoto University, Kyoto 606-8501, Japan.
Knut
K
Woltjen
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.
Ian
I
Chambers
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, Scotland. Electronic address: keisuke.kaji@ed.ac.uk.
Keisuke
K
Kaji
D000818:Animals; D065150:Cellular Reprogramming; D058750:Epithelial-Mesenchymal Transition; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000071317:Nanog Homeobox Protein
D018398:Homeodomain Proteins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse