SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

Marta Winiecka-Klimek; Maciej Smolarz; Maciej P Walczak; Jolanta Zieba; Krystyna Hulas-Bigoszewska; Blazej Kmieciak; Sylwester Piaskowski; Piotr Rieske; Dawid P Grzela; Ewelina Stoczynska-Fidelus

doi:10.1371/journal.pone.0141688

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PLoS One. 2015 Nov 4;10(11):e0141688. doi: 10.1371/journal.pone.0141688. eCollection 2015.

Authors

Marta Winiecka-Klimek^{1

2}, Maciej Smolarz^{1

2}, Maciej P Walczak¹, Jolanta Zieba^{1

2}, Krystyna Hulas-Bigoszewska¹, Blazej Kmieciak³, Sylwester Piaskowski^{1

2}, Piotr Rieske^{1

2}, Dawid P Grzela¹, Ewelina Stoczynska-Fidelus^{1

2}

Affiliations

¹ Department of Research and Development, Celther Polska, Lodz, Poland.
² Department of Tumor Biology, Medical University of Lodz, Lodz, Poland.
³ Department of Medical Law, Chair of Human Sciences, Medical University of Lodz, Lodz, Poland.

Abstract

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods-direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc-in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cellular Reprogramming Techniques / methods
Cellular Reprogramming*
Cellular Senescence*
Fibroblasts / metabolism*
Humans
Neural Stem Cells / metabolism*
Proto-Oncogene Proteins c-myc / biosynthesis*
Proto-Oncogene Proteins c-myc / genetics
SOXB1 Transcription Factors / biosynthesis*
SOXB1 Transcription Factors / genetics
Transduction, Genetic / methods

Substances

MYC protein, human
Proto-Oncogene Proteins c-myc
SOX2 protein, human
SOXB1 Transcription Factors

Grants and funding

This study was sponsored by Polish Agency for Enterprise Development, Grant No. POIG.01.04.00-10-011/11. ESF, PR, and SP participated in acquisition of funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.