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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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Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4, Klf4, Sox2, c-Myc; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.
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Figure 1: Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4, Klf4, Sox2, c-Myc; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.

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)

Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4, Klf4, Sox2, c-Myc; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4326224&req=5

Figure 1: Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4, Klf4, Sox2, c-Myc; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.
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