Limits...
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

Cellular and molecular characterization of iPS clones derived by Sox2 protein transduction into OKC-MEFs. (a) Pictures of isolated cell lines Sox2-piPS-1 (upper row) and Sox2-piPS-2 (lower row) exhibiting brightfield (BF), staining against pluripotency-associated marker SSEA-1 and native GFP fluorescence. Sox2-piPS-1 cell line was clonally isolated from 400 nM Sox2-TAT treatment from day 1 to 5, and Sox2-piPS-2 was derived from 200 nM condition (day 5 to 10) Scale bar = 100 μm. (b) PCR analysis of genomic DNA demonstrating genomic integration of Oct4 and Klf4 transgenes. As expected, no transgenic Sox2 was detected in Sox2-piPS clones excluding possibility of contamination. (c) RT-PCR analysis demonstrating transgene silencing in Sox2-piPS cells. Primers specific for transgenic Oct4, Sox2, Klf4, and c-Myc were used. Additionally, we analyzed endogenous Oct4, Sox2, and Nanog transcripts. RNA preparations from infected (Inf.) and uninfected MEFs as well as ES cells served as controls.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368608&req=5

fig4: Cellular and molecular characterization of iPS clones derived by Sox2 protein transduction into OKC-MEFs. (a) Pictures of isolated cell lines Sox2-piPS-1 (upper row) and Sox2-piPS-2 (lower row) exhibiting brightfield (BF), staining against pluripotency-associated marker SSEA-1 and native GFP fluorescence. Sox2-piPS-1 cell line was clonally isolated from 400 nM Sox2-TAT treatment from day 1 to 5, and Sox2-piPS-2 was derived from 200 nM condition (day 5 to 10) Scale bar = 100 μm. (b) PCR analysis of genomic DNA demonstrating genomic integration of Oct4 and Klf4 transgenes. As expected, no transgenic Sox2 was detected in Sox2-piPS clones excluding possibility of contamination. (c) RT-PCR analysis demonstrating transgene silencing in Sox2-piPS cells. Primers specific for transgenic Oct4, Sox2, Klf4, and c-Myc were used. Additionally, we analyzed endogenous Oct4, Sox2, and Nanog transcripts. RNA preparations from infected (Inf.) and uninfected MEFs as well as ES cells served as controls.

Mentions: Two Sox2-protein iPS colonies were isolated and expanded for further characterization, yielding Sox2-piPS-1 and Sox2-piPS-2 cell lines, respectively. Both could be proliferated for at least 20 passages, and they maintained their Oct4 promoter-driven GFP activity. Moreover, they stained positively for the pluripotency-associated cell surface marker SSEA-1 (Figure 4(a)). Sox2-piPS-1 and Sox2-piPS-2 were subjected to PCR analysis in order to assess transgenic integrations. This analysis revealed that both lines carry integrated viral transgenes, but no exogenous Sox2 (Figure 4(b)). Transgene silencing represents a major hallmark of successful iPS derivation. Thus, we applied RT-PCR analysis to detect the transcripts of the transgenic reprogramming factors as well as endogenous stemness factors. Both clones analyzed exhibit no detectable transgenic Oct4, Sox2, Klf4, or c-Myc (Figure 4(c)). The mRNA of endogenous Oct4, Sox2 and Nanog, in contrast, was found as abundant as in the ES control cells, indicating complete silencing of exogenous factors and reactivation of the endogenous grid of stemness. Finally, we set out to confirm the pluripotent status of Sox2-piPS-2 by spontaneous differentiation into embryoid bodies (EBs). 5-day old EBs were plated and analyzed for the appearance of specific germ layer marker by staining against β-3-tubulin (TUJ1), smooth muscle actin (SMA), and α-feto-protein (AFP) (Figure 5). According to this analysis, Sox2-piPS-2 cells differentiated into all three germ layers, demonstrating an unrestricted in vitro differentiation potential.


Cellular reprogramming employing recombinant sox2 protein.

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

Cellular and molecular characterization of iPS clones derived by Sox2 protein transduction into OKC-MEFs. (a) Pictures of isolated cell lines Sox2-piPS-1 (upper row) and Sox2-piPS-2 (lower row) exhibiting brightfield (BF), staining against pluripotency-associated marker SSEA-1 and native GFP fluorescence. Sox2-piPS-1 cell line was clonally isolated from 400 nM Sox2-TAT treatment from day 1 to 5, and Sox2-piPS-2 was derived from 200 nM condition (day 5 to 10) Scale bar = 100 μm. (b) PCR analysis of genomic DNA demonstrating genomic integration of Oct4 and Klf4 transgenes. As expected, no transgenic Sox2 was detected in Sox2-piPS clones excluding possibility of contamination. (c) RT-PCR analysis demonstrating transgene silencing in Sox2-piPS cells. Primers specific for transgenic Oct4, Sox2, Klf4, and c-Myc were used. Additionally, we analyzed endogenous Oct4, Sox2, and Nanog transcripts. RNA preparations from infected (Inf.) and uninfected MEFs as well as ES cells served as controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Cellular and molecular characterization of iPS clones derived by Sox2 protein transduction into OKC-MEFs. (a) Pictures of isolated cell lines Sox2-piPS-1 (upper row) and Sox2-piPS-2 (lower row) exhibiting brightfield (BF), staining against pluripotency-associated marker SSEA-1 and native GFP fluorescence. Sox2-piPS-1 cell line was clonally isolated from 400 nM Sox2-TAT treatment from day 1 to 5, and Sox2-piPS-2 was derived from 200 nM condition (day 5 to 10) Scale bar = 100 μm. (b) PCR analysis of genomic DNA demonstrating genomic integration of Oct4 and Klf4 transgenes. As expected, no transgenic Sox2 was detected in Sox2-piPS clones excluding possibility of contamination. (c) RT-PCR analysis demonstrating transgene silencing in Sox2-piPS cells. Primers specific for transgenic Oct4, Sox2, Klf4, and c-Myc were used. Additionally, we analyzed endogenous Oct4, Sox2, and Nanog transcripts. RNA preparations from infected (Inf.) and uninfected MEFs as well as ES cells served as controls.
Mentions: Two Sox2-protein iPS colonies were isolated and expanded for further characterization, yielding Sox2-piPS-1 and Sox2-piPS-2 cell lines, respectively. Both could be proliferated for at least 20 passages, and they maintained their Oct4 promoter-driven GFP activity. Moreover, they stained positively for the pluripotency-associated cell surface marker SSEA-1 (Figure 4(a)). Sox2-piPS-1 and Sox2-piPS-2 were subjected to PCR analysis in order to assess transgenic integrations. This analysis revealed that both lines carry integrated viral transgenes, but no exogenous Sox2 (Figure 4(b)). Transgene silencing represents a major hallmark of successful iPS derivation. Thus, we applied RT-PCR analysis to detect the transcripts of the transgenic reprogramming factors as well as endogenous stemness factors. Both clones analyzed exhibit no detectable transgenic Oct4, Sox2, Klf4, or c-Myc (Figure 4(c)). The mRNA of endogenous Oct4, Sox2 and Nanog, in contrast, was found as abundant as in the ES control cells, indicating complete silencing of exogenous factors and reactivation of the endogenous grid of stemness. Finally, we set out to confirm the pluripotent status of Sox2-piPS-2 by spontaneous differentiation into embryoid bodies (EBs). 5-day old EBs were plated and analyzed for the appearance of specific germ layer marker by staining against β-3-tubulin (TUJ1), smooth muscle actin (SMA), and α-feto-protein (AFP) (Figure 5). According to this analysis, Sox2-piPS-2 cells differentiated into all three germ layers, demonstrating an unrestricted in vitro differentiation potential.

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