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Small molecules facilitate rapid and synchronous iPSC generation.

Bar-Nur O, Brumbaugh J, Verheul C, Apostolou E, Pruteanu-Malinici I, Walsh RM, Ramaswamy S, Hochedlinger K - Nat. Methods (2014)

Bottom Line: However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations.Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression.Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

View Article: PubMed Central - PubMed

Affiliation: 1] Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [4] Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA.

ABSTRACT
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

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AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published43. (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).
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Figure 2: AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published43. (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).

Mentions: Studying the process of cellular reprogramming with classical tools has been hampered by the inability to monitor exogenous OKSM expression patterns in somatic cells. We therefore generated a transgenic reprogramming system in mice that allowed us to simultaneously induce and track high-level OKSM expression in any target cells (Fig. 1a). To this end, mice homozygous for the doxycycline-inducible, polycistronic tetOP-OKSM construct in the Col1a1 locus15 were crossed to mice homozygous for a cassette containing the coding regions for Oct4, Klf4, Sox2 and an IRES-mCherry reporter in the Col1a1 locus (tetOP-OKSmC)(data not shown) and the M2rtTA allele in the Rosa26 locus (R26-M2rtTA)15. Doxycycline treatment of murine embryonic fibroblasts (MEFs) isolated from this cross induced strong and homogeneous expression of reprogramming factors, as determined by microscopy for mCherry (Fig. 1b), and consistently gave rise to iPSCs from different cell types under conventional culture conditions (Fig. 1c–f, Fig. 2a). Cells carrying the tetOP-OKSM, tetOP-OKSmC and R26-M2rtTA alleles were used for all subsequent experiments unless noted otherwise.


Small molecules facilitate rapid and synchronous iPSC generation.

Bar-Nur O, Brumbaugh J, Verheul C, Apostolou E, Pruteanu-Malinici I, Walsh RM, Ramaswamy S, Hochedlinger K - Nat. Methods (2014)

AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published43. (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4326224&req=5

Figure 2: AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published43. (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).
Mentions: Studying the process of cellular reprogramming with classical tools has been hampered by the inability to monitor exogenous OKSM expression patterns in somatic cells. We therefore generated a transgenic reprogramming system in mice that allowed us to simultaneously induce and track high-level OKSM expression in any target cells (Fig. 1a). To this end, mice homozygous for the doxycycline-inducible, polycistronic tetOP-OKSM construct in the Col1a1 locus15 were crossed to mice homozygous for a cassette containing the coding regions for Oct4, Klf4, Sox2 and an IRES-mCherry reporter in the Col1a1 locus (tetOP-OKSmC)(data not shown) and the M2rtTA allele in the Rosa26 locus (R26-M2rtTA)15. Doxycycline treatment of murine embryonic fibroblasts (MEFs) isolated from this cross induced strong and homogeneous expression of reprogramming factors, as determined by microscopy for mCherry (Fig. 1b), and consistently gave rise to iPSCs from different cell types under conventional culture conditions (Fig. 1c–f, Fig. 2a). Cells carrying the tetOP-OKSM, tetOP-OKSmC and R26-M2rtTA alleles were used for all subsequent experiments unless noted otherwise.

Bottom Line: However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations.Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression.Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

View Article: PubMed Central - PubMed

Affiliation: 1] Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [4] Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA.

ABSTRACT
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

Show MeSH
Related in: MedlinePlus