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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

Purification of recombinant Sox2-TAT fusion protein and reprogramming setup. (a) The recombinant cell-permeant Sox2 fusion protein [22] consists of the full-length Sox2 protein and a carboxy-terminally fused sequence of tags consisting of a nuclear localization sequence (NLS), cell-penetrating peptide TAT, and a histidine-tag (H6) for single-step purification. (b) Schematic representation of the expression and purification procedure and the reprogramming setup used in this study. After expression in E. coli, Sox2-TAT-containing cell lysates are subjected to affinity column chromatography employing Ni-NTA resin. Purified recombinant Sox2-TAT protein is eluted from the column and its reprogramming competency assessed in combination with retroviruses encoding Oct4, Klf-4, and c-Myc. (c, d) Biochemical analysis of Sox2-TAT purification from E. coli. The following fractions were subjected to SDS-PAGE analysis: crude lysate (CL), marker (M), pellet (P), supernatant (SN), flow-through (FT), washing buffer (W), and elution fraction (E). SDS gels were either stained using Coomassie (c) or used for preparation of an immunoblot using anti-Sox2-specific antibody (d).
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fig1: Purification of recombinant Sox2-TAT fusion protein and reprogramming setup. (a) The recombinant cell-permeant Sox2 fusion protein [22] consists of the full-length Sox2 protein and a carboxy-terminally fused sequence of tags consisting of a nuclear localization sequence (NLS), cell-penetrating peptide TAT, and a histidine-tag (H6) for single-step purification. (b) Schematic representation of the expression and purification procedure and the reprogramming setup used in this study. After expression in E. coli, Sox2-TAT-containing cell lysates are subjected to affinity column chromatography employing Ni-NTA resin. Purified recombinant Sox2-TAT protein is eluted from the column and its reprogramming competency assessed in combination with retroviruses encoding Oct4, Klf-4, and c-Myc. (c, d) Biochemical analysis of Sox2-TAT purification from E. coli. The following fractions were subjected to SDS-PAGE analysis: crude lysate (CL), marker (M), pellet (P), supernatant (SN), flow-through (FT), washing buffer (W), and elution fraction (E). SDS gels were either stained using Coomassie (c) or used for preparation of an immunoblot using anti-Sox2-specific antibody (d).

Mentions: We have previously shown that recombinant Sox2 can be purified from E. coli as a TAT-modified cell-permeant version, designated Sox2-TAT [22]. In particular, this fusion protein comprises an additional exogenous nuclear localization sequence (NLS), a cell-penetrating peptide TAT, and a carboxy-terminal Histidine-tag for single-step purification (Figure 1(a)). Sox2-TAT was shown to specifically bind to DNA and to compensate for the RNAi-induced loss of activity in ES cells [22] and preimplantation embryos [32]; however, its capability to reprogram somatic cells has not been assessed. In order to study the reprogramming activity of Sox2-TAT, we decided to combine the purified recombinant Sox2-TAT together with retroviruses encoding for Oct4, Klf4, and c-Myc to convert mouse embryonic fibroblasts (MEFs) into iPS cells (Figure 1(b)). Sox2-TAT-transformed bacteria were lysed and subjected to Ni-affinity chromatography. Immunoblotting of purification fractions employing a His-specific antibody revealed that Sox2-TAT is highly expressed in bacteria although the majority of the recombinant protein remains in the insoluble fraction (Figure 1(d)). However, the estimated 20% of protein solubilized and detectable in the supernatant turned out to be sufficient for further purification. The elution from the Ni-affinity chromatography column yielded a Sox2-TAT-containing fraction of about 70% purity (Figure 1(c)).


Cellular reprogramming employing recombinant sox2 protein.

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

Purification of recombinant Sox2-TAT fusion protein and reprogramming setup. (a) The recombinant cell-permeant Sox2 fusion protein [22] consists of the full-length Sox2 protein and a carboxy-terminally fused sequence of tags consisting of a nuclear localization sequence (NLS), cell-penetrating peptide TAT, and a histidine-tag (H6) for single-step purification. (b) Schematic representation of the expression and purification procedure and the reprogramming setup used in this study. After expression in E. coli, Sox2-TAT-containing cell lysates are subjected to affinity column chromatography employing Ni-NTA resin. Purified recombinant Sox2-TAT protein is eluted from the column and its reprogramming competency assessed in combination with retroviruses encoding Oct4, Klf-4, and c-Myc. (c, d) Biochemical analysis of Sox2-TAT purification from E. coli. The following fractions were subjected to SDS-PAGE analysis: crude lysate (CL), marker (M), pellet (P), supernatant (SN), flow-through (FT), washing buffer (W), and elution fraction (E). SDS gels were either stained using Coomassie (c) or used for preparation of an immunoblot using anti-Sox2-specific antibody (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Purification of recombinant Sox2-TAT fusion protein and reprogramming setup. (a) The recombinant cell-permeant Sox2 fusion protein [22] consists of the full-length Sox2 protein and a carboxy-terminally fused sequence of tags consisting of a nuclear localization sequence (NLS), cell-penetrating peptide TAT, and a histidine-tag (H6) for single-step purification. (b) Schematic representation of the expression and purification procedure and the reprogramming setup used in this study. After expression in E. coli, Sox2-TAT-containing cell lysates are subjected to affinity column chromatography employing Ni-NTA resin. Purified recombinant Sox2-TAT protein is eluted from the column and its reprogramming competency assessed in combination with retroviruses encoding Oct4, Klf-4, and c-Myc. (c, d) Biochemical analysis of Sox2-TAT purification from E. coli. The following fractions were subjected to SDS-PAGE analysis: crude lysate (CL), marker (M), pellet (P), supernatant (SN), flow-through (FT), washing buffer (W), and elution fraction (E). SDS gels were either stained using Coomassie (c) or used for preparation of an immunoblot using anti-Sox2-specific antibody (d).
Mentions: We have previously shown that recombinant Sox2 can be purified from E. coli as a TAT-modified cell-permeant version, designated Sox2-TAT [22]. In particular, this fusion protein comprises an additional exogenous nuclear localization sequence (NLS), a cell-penetrating peptide TAT, and a carboxy-terminal Histidine-tag for single-step purification (Figure 1(a)). Sox2-TAT was shown to specifically bind to DNA and to compensate for the RNAi-induced loss of activity in ES cells [22] and preimplantation embryos [32]; however, its capability to reprogram somatic cells has not been assessed. In order to study the reprogramming activity of Sox2-TAT, we decided to combine the purified recombinant Sox2-TAT together with retroviruses encoding for Oct4, Klf4, and c-Myc to convert mouse embryonic fibroblasts (MEFs) into iPS cells (Figure 1(b)). Sox2-TAT-transformed bacteria were lysed and subjected to Ni-affinity chromatography. Immunoblotting of purification fractions employing a His-specific antibody revealed that Sox2-TAT is highly expressed in bacteria although the majority of the recombinant protein remains in the insoluble fraction (Figure 1(d)). However, the estimated 20% of protein solubilized and detectable in the supernatant turned out to be sufficient for further purification. The elution from the Ni-affinity chromatography column yielded a Sox2-TAT-containing fraction of about 70% purity (Figure 1(c)).

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