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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

Effect of media supplements on the stability of cell-permeant Sox2-TAT fusion protein and efficiency of protein delivery. (a) Ability of media supplements to stabilize Sox2-TAT. Fetal calf serum (FCS), Albumax, and serum replacement (SR) were added to the eluate fraction and subsequently dialyzed against DMEM-F12 media. Depicted are anti-Sox2-immunoblots of the dialysis fraction (D) and a stability test of samples being taken after 1 hour (1 h) and 6 hours (6 h), respectively. (b) Influence of FCS, SR, and a combination of both on the transduction efficiency. Protein transduction efficiencies were analyzed by quantifying the recombined cells after delivery of cell permeant Cre-protein (TAT-Cre) into the CV1-5B Cre reporter cell line. Cells were treated with different concentrations of TAT-Cre (0.25 μM–2 μM) in transduction media supplemented with either 15% serum replacement, 5% FCS, or mixture of 2% FCS and 7.5% SR. To determine the recombination activity, cells were fixed and stained for β-galactosidase activity after 48 hours. Cre protein transduction and quantification of recombination in Cre reporter cells was performed as described previously [35].
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fig2: Effect of media supplements on the stability of cell-permeant Sox2-TAT fusion protein and efficiency of protein delivery. (a) Ability of media supplements to stabilize Sox2-TAT. Fetal calf serum (FCS), Albumax, and serum replacement (SR) were added to the eluate fraction and subsequently dialyzed against DMEM-F12 media. Depicted are anti-Sox2-immunoblots of the dialysis fraction (D) and a stability test of samples being taken after 1 hour (1 h) and 6 hours (6 h), respectively. (b) Influence of FCS, SR, and a combination of both on the transduction efficiency. Protein transduction efficiencies were analyzed by quantifying the recombined cells after delivery of cell permeant Cre-protein (TAT-Cre) into the CV1-5B Cre reporter cell line. Cells were treated with different concentrations of TAT-Cre (0.25 μM–2 μM) in transduction media supplemented with either 15% serum replacement, 5% FCS, or mixture of 2% FCS and 7.5% SR. To determine the recombination activity, cells were fixed and stained for β-galactosidase activity after 48 hours. Cre protein transduction and quantification of recombination in Cre reporter cells was performed as described previously [35].

Mentions: Poor solubility and limited stability of recombinant proteins under cell culture conditions represent a significant hurdle to the application of protein transduction technology. On the one hand, serum components stabilize recombinant proteins in cell culture media, but on the other hand, they are known to inhibit interaction of transducible proteins with cells and by this decrease the cellular uptake. In some experimental settings, this can be overcome by applying the transducible protein in serum-free media. We assessed the stability of Sox2-TAT employing various cell culture conditions. In serum-free media, Sox2-TAT precipitates almost completely within 1 hour (Figure 2(a)). Serum components have been shown to execute a positive effect on the stability of recombinant proteins [36, 40]. Therefore, we aimed at stabilizing the protein by supplements like FCS, serum replacement [35], and lipid-rich albumin fractions (Albumax). Supplementation with either 5% FCS or 2.5% Albumax showed a strong stabilizing effect on Sox2-TAT in culture media, while Sox2-TAT exhibited major decrease in the presence of 7.5% SR. A combination of low FCS (2%) together with 7.5% SR resulted in a stabilization that was comparable to high FCS supplementation (5%) (Figure 2(a)).


Cellular reprogramming employing recombinant sox2 protein.

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

Effect of media supplements on the stability of cell-permeant Sox2-TAT fusion protein and efficiency of protein delivery. (a) Ability of media supplements to stabilize Sox2-TAT. Fetal calf serum (FCS), Albumax, and serum replacement (SR) were added to the eluate fraction and subsequently dialyzed against DMEM-F12 media. Depicted are anti-Sox2-immunoblots of the dialysis fraction (D) and a stability test of samples being taken after 1 hour (1 h) and 6 hours (6 h), respectively. (b) Influence of FCS, SR, and a combination of both on the transduction efficiency. Protein transduction efficiencies were analyzed by quantifying the recombined cells after delivery of cell permeant Cre-protein (TAT-Cre) into the CV1-5B Cre reporter cell line. Cells were treated with different concentrations of TAT-Cre (0.25 μM–2 μM) in transduction media supplemented with either 15% serum replacement, 5% FCS, or mixture of 2% FCS and 7.5% SR. To determine the recombination activity, cells were fixed and stained for β-galactosidase activity after 48 hours. Cre protein transduction and quantification of recombination in Cre reporter cells was performed as described previously [35].
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368608&req=5

fig2: Effect of media supplements on the stability of cell-permeant Sox2-TAT fusion protein and efficiency of protein delivery. (a) Ability of media supplements to stabilize Sox2-TAT. Fetal calf serum (FCS), Albumax, and serum replacement (SR) were added to the eluate fraction and subsequently dialyzed against DMEM-F12 media. Depicted are anti-Sox2-immunoblots of the dialysis fraction (D) and a stability test of samples being taken after 1 hour (1 h) and 6 hours (6 h), respectively. (b) Influence of FCS, SR, and a combination of both on the transduction efficiency. Protein transduction efficiencies were analyzed by quantifying the recombined cells after delivery of cell permeant Cre-protein (TAT-Cre) into the CV1-5B Cre reporter cell line. Cells were treated with different concentrations of TAT-Cre (0.25 μM–2 μM) in transduction media supplemented with either 15% serum replacement, 5% FCS, or mixture of 2% FCS and 7.5% SR. To determine the recombination activity, cells were fixed and stained for β-galactosidase activity after 48 hours. Cre protein transduction and quantification of recombination in Cre reporter cells was performed as described previously [35].
Mentions: Poor solubility and limited stability of recombinant proteins under cell culture conditions represent a significant hurdle to the application of protein transduction technology. On the one hand, serum components stabilize recombinant proteins in cell culture media, but on the other hand, they are known to inhibit interaction of transducible proteins with cells and by this decrease the cellular uptake. In some experimental settings, this can be overcome by applying the transducible protein in serum-free media. We assessed the stability of Sox2-TAT employing various cell culture conditions. In serum-free media, Sox2-TAT precipitates almost completely within 1 hour (Figure 2(a)). Serum components have been shown to execute a positive effect on the stability of recombinant proteins [36, 40]. Therefore, we aimed at stabilizing the protein by supplements like FCS, serum replacement [35], and lipid-rich albumin fractions (Albumax). Supplementation with either 5% FCS or 2.5% Albumax showed a strong stabilizing effect on Sox2-TAT in culture media, while Sox2-TAT exhibited major decrease in the presence of 7.5% SR. A combination of low FCS (2%) together with 7.5% SR resulted in a stabilization that was comparable to high FCS supplementation (5%) (Figure 2(a)).

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