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Cellular reprogramming employing recombinant sox2 protein.

Thier M, Münst B, Mielke S, Edenhofer F - Stem Cells Int (2012)

Bottom Line: To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes.Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2.Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn-Life & Brain Center and Hertie Foundation, Sigmund-Freud Straße 25, D-53105 Bonn, Germany.

ABSTRACT
Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

No MeSH data available.


Related in: MedlinePlus

Reprogramming of MEFs using cell-permeant Sox2-TAT protein. (a) Schematic presentation showing the timeline of reprogramming setup. Oct4-GiP MEFs were infected with viruses encoding Oct4, Klf4, and c-Myc (OKC) at day 0. Starting at day 1 post infection (p.i.), cells were incubated with Sox2-TAT for 5 and 10 days, respectively, changing the Sox2-TAT-supplemented media daily. (b) Representative pictures of cells transduced with 200 nM Sox2-TAT protein (right panel) displaying phase contrast and GFP channel (inset) 14 days p.i. OKC-infected cells treated with medium only served as controls (left panel). Scale bar = 100 μm. (c) Quantification of GFP-positive colonies at day 16 p.i. OKC-infected cells (−) as well as SOKC-infected cells (+) served as controls.
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fig3: Reprogramming of MEFs using cell-permeant Sox2-TAT protein. (a) Schematic presentation showing the timeline of reprogramming setup. Oct4-GiP MEFs were infected with viruses encoding Oct4, Klf4, and c-Myc (OKC) at day 0. Starting at day 1 post infection (p.i.), cells were incubated with Sox2-TAT for 5 and 10 days, respectively, changing the Sox2-TAT-supplemented media daily. (b) Representative pictures of cells transduced with 200 nM Sox2-TAT protein (right panel) displaying phase contrast and GFP channel (inset) 14 days p.i. OKC-infected cells treated with medium only served as controls (left panel). Scale bar = 100 μm. (c) Quantification of GFP-positive colonies at day 16 p.i. OKC-infected cells (−) as well as SOKC-infected cells (+) served as controls.

Mentions: We then assessed the reprogramming activity of Sox2-TAT employing the optimized media conditions. For that, we used a modification of the classical four-factor viral reprogramming paradigm and aimed at substituting the Sox2-encoding virus by Sox2-TAT protein (Figure 3(a)). We transfected Oct4-GiP-transgenic MEFs [38], which enable GFP-based monitoring of reprogramming by reactivation of the Oct4 promoter, with viruses encoding for Oct-4, Klf4, and c-Myc (OKC). OKC-MEFs were initially cultivated for five days in medium containing either 200 nM or 400 nM Sox2-TAT. During the whole reprogramming procedure, we changed the protein-supplemented media every day to ensure a continuous delivery of the recombinant reprogramming factor. At day five, cells were split onto freshly plated feeder cells and either cultured in normal media or further exposed to Sox2-TAT-containing media for five more days (Figure 3(a)). First, iPS-like structures appeared after 9 days and formed well-defined GFP-positive colonies (Figure 3(b)). The viral transduction of the three factors Oct4, Klf4, and c-Myc without Sox2-TAT protein did not yield any GFP-positive colony (Figure 3(b)). GFP+ colonies were quantified at day 16. We counted eight GFP+ colonies in wells containing cells treated with 400 nM Sox2-TAT for five days. Prolonged incubation with Sox2-TAT until day 10 did not result in a marked increase of colony numbers. Instead, the number of colonies slightly decreased eventually due a strict time window required for Sox2 application. The application of 200 nM Sox2-TAT for five and ten days yielded no and just one GFP+ colony, respectively (Figure 3(c)), indicating that the Sox2-TAT concentration is a limiting factor. We aimed at further increasing the Sox2-TAT concentration by either dialysis against glycerol-containing concentration buffer or ultrafiltration; however, beyond 400 nM, the protein strongly precipitated in culture media and interfering with cellular growth (data not shown).


Cellular reprogramming employing recombinant sox2 protein.

Thier M, Münst B, Mielke S, Edenhofer F - Stem Cells Int (2012)

Reprogramming of MEFs using cell-permeant Sox2-TAT protein. (a) Schematic presentation showing the timeline of reprogramming setup. Oct4-GiP MEFs were infected with viruses encoding Oct4, Klf4, and c-Myc (OKC) at day 0. Starting at day 1 post infection (p.i.), cells were incubated with Sox2-TAT for 5 and 10 days, respectively, changing the Sox2-TAT-supplemented media daily. (b) Representative pictures of cells transduced with 200 nM Sox2-TAT protein (right panel) displaying phase contrast and GFP channel (inset) 14 days p.i. OKC-infected cells treated with medium only served as controls (left panel). Scale bar = 100 μm. (c) Quantification of GFP-positive colonies at day 16 p.i. OKC-infected cells (−) as well as SOKC-infected cells (+) served as controls.
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Related In: Results  -  Collection

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fig3: Reprogramming of MEFs using cell-permeant Sox2-TAT protein. (a) Schematic presentation showing the timeline of reprogramming setup. Oct4-GiP MEFs were infected with viruses encoding Oct4, Klf4, and c-Myc (OKC) at day 0. Starting at day 1 post infection (p.i.), cells were incubated with Sox2-TAT for 5 and 10 days, respectively, changing the Sox2-TAT-supplemented media daily. (b) Representative pictures of cells transduced with 200 nM Sox2-TAT protein (right panel) displaying phase contrast and GFP channel (inset) 14 days p.i. OKC-infected cells treated with medium only served as controls (left panel). Scale bar = 100 μm. (c) Quantification of GFP-positive colonies at day 16 p.i. OKC-infected cells (−) as well as SOKC-infected cells (+) served as controls.
Mentions: We then assessed the reprogramming activity of Sox2-TAT employing the optimized media conditions. For that, we used a modification of the classical four-factor viral reprogramming paradigm and aimed at substituting the Sox2-encoding virus by Sox2-TAT protein (Figure 3(a)). We transfected Oct4-GiP-transgenic MEFs [38], which enable GFP-based monitoring of reprogramming by reactivation of the Oct4 promoter, with viruses encoding for Oct-4, Klf4, and c-Myc (OKC). OKC-MEFs were initially cultivated for five days in medium containing either 200 nM or 400 nM Sox2-TAT. During the whole reprogramming procedure, we changed the protein-supplemented media every day to ensure a continuous delivery of the recombinant reprogramming factor. At day five, cells were split onto freshly plated feeder cells and either cultured in normal media or further exposed to Sox2-TAT-containing media for five more days (Figure 3(a)). First, iPS-like structures appeared after 9 days and formed well-defined GFP-positive colonies (Figure 3(b)). The viral transduction of the three factors Oct4, Klf4, and c-Myc without Sox2-TAT protein did not yield any GFP-positive colony (Figure 3(b)). GFP+ colonies were quantified at day 16. We counted eight GFP+ colonies in wells containing cells treated with 400 nM Sox2-TAT for five days. Prolonged incubation with Sox2-TAT until day 10 did not result in a marked increase of colony numbers. Instead, the number of colonies slightly decreased eventually due a strict time window required for Sox2 application. The application of 200 nM Sox2-TAT for five and ten days yielded no and just one GFP+ colony, respectively (Figure 3(c)), indicating that the Sox2-TAT concentration is a limiting factor. We aimed at further increasing the Sox2-TAT concentration by either dialysis against glycerol-containing concentration buffer or ultrafiltration; however, beyond 400 nM, the protein strongly precipitated in culture media and interfering with cellular growth (data not shown).

Bottom Line: To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes.Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2.Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn-Life & Brain Center and Hertie Foundation, Sigmund-Freud Straße 25, D-53105 Bonn, Germany.

ABSTRACT
Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

No MeSH data available.


Related in: MedlinePlus