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

Winiecka-Klimek M, Smolarz M, Walczak MP, Zieba J, Hulas-Bigoszewska K, Kmieciak B, Piaskowski S, Rieske P, Grzela DP, Stoczynska-Fidelus E - PLoS ONE (2015)

Bottom Line: Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Research and Development, Celther Polska, 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.

No MeSH data available.


Related in: MedlinePlus

Immunocytochemical detection of SOX2 expression four days after transduction.BJ fibroblasts were stained with anti-SOX2 antibody (A), counterstained with 4’,6-diamidino-2-phenylindole, DAPI (B) and results of staining were merged (C). Images were captured using Eclipse Ci-S epifluorescence microscope with 40x objective; scale bar = 50 μm.
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pone.0141688.g001: Immunocytochemical detection of SOX2 expression four days after transduction.BJ fibroblasts were stained with anti-SOX2 antibody (A), counterstained with 4’,6-diamidino-2-phenylindole, DAPI (B) and results of staining were merged (C). Images were captured using Eclipse Ci-S epifluorescence microscope with 40x objective; scale bar = 50 μm.

Mentions: To assess the transduction efficiency, four days after lentiviral infection, immunocytochemical staining for SOX2 expression was performed. All cells were SOX2-positive indicating high transduction efficiency of the lentiviral vector (Fig 1). Nevertheless, after 36 days in culture, substantial repression of SOX2 expression was detected, as only single SOX2-positive cells were observed.


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

Winiecka-Klimek M, Smolarz M, Walczak MP, Zieba J, Hulas-Bigoszewska K, Kmieciak B, Piaskowski S, Rieske P, Grzela DP, Stoczynska-Fidelus E - PLoS ONE (2015)

Immunocytochemical detection of SOX2 expression four days after transduction.BJ fibroblasts were stained with anti-SOX2 antibody (A), counterstained with 4’,6-diamidino-2-phenylindole, DAPI (B) and results of staining were merged (C). Images were captured using Eclipse Ci-S epifluorescence microscope with 40x objective; scale bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4633175&req=5

pone.0141688.g001: Immunocytochemical detection of SOX2 expression four days after transduction.BJ fibroblasts were stained with anti-SOX2 antibody (A), counterstained with 4’,6-diamidino-2-phenylindole, DAPI (B) and results of staining were merged (C). Images were captured using Eclipse Ci-S epifluorescence microscope with 40x objective; scale bar = 50 μm.
Mentions: To assess the transduction efficiency, four days after lentiviral infection, immunocytochemical staining for SOX2 expression was performed. All cells were SOX2-positive indicating high transduction efficiency of the lentiviral vector (Fig 1). Nevertheless, after 36 days in culture, substantial repression of SOX2 expression was detected, as only single SOX2-positive cells were observed.

Bottom Line: Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Research and Development, Celther Polska, 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.

No MeSH data available.


Related in: MedlinePlus