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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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Effect of AGi on gene expression patterns in reprogramming intermediates. (a) Unsupervised clustering of global gene expression analysis of indicated samples. “4–12 days OKSM” represent reprogrammable MEFs exposed for 4–12 days to doxycycline in the absence or presence of AGi. (b) A heat map showing expression levels for genes that were greater than three-fold higher in ESCs relative to MEFs. (c) DAVID functional analysis of genes whose expression is downregulated at least 1.5 fold in response to AGi treatment in MEFs expressing OKSM/OKSM+AGi. Note the prevalence of developmental regulators. A Benjamini-Hochberg (BH) adjusted p-value is presented. (d) Examples of somatic, pluripotency, microRNA, and transient developmental genes that change expression in response to doxycycline/AGi treatment. (e) Expression patterns of gene sets associated with a refractory phenotype (left panel) or transient upregulation during reprogramming (right panel) were compared with expression patterns obtained after OKSM expression in the presence or absence of AGi. Statistical significance is indicated with asterisks (One-tailed Fischer test).
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Figure 5: Effect of AGi on gene expression patterns in reprogramming intermediates. (a) Unsupervised clustering of global gene expression analysis of indicated samples. “4–12 days OKSM” represent reprogrammable MEFs exposed for 4–12 days to doxycycline in the absence or presence of AGi. (b) A heat map showing expression levels for genes that were greater than three-fold higher in ESCs relative to MEFs. (c) DAVID functional analysis of genes whose expression is downregulated at least 1.5 fold in response to AGi treatment in MEFs expressing OKSM/OKSM+AGi. Note the prevalence of developmental regulators. A Benjamini-Hochberg (BH) adjusted p-value is presented. (d) Examples of somatic, pluripotency, microRNA, and transient developmental genes that change expression in response to doxycycline/AGi treatment. (e) Expression patterns of gene sets associated with a refractory phenotype (left panel) or transient upregulation during reprogramming (right panel) were compared with expression patterns obtained after OKSM expression in the presence or absence of AGi. Statistical significance is indicated with asterisks (One-tailed Fischer test).

Mentions: Successful reprogramming requires extinction of the somatic program and activation of key pluripotency factors7. Furthermore, cell populations expressing OKSM were reported to transiently upregulate developmental genes10,28; however, the relevance of these changes remains unclear. In an attempt to dissect the molecular consequences of AGi treatment, we performed expression profiling of reprogrammable MEFs exposed to doxycycline for 4, 6, 8, 10, 12 days in the presence or absence of AGi. Unsupervised clustering of the entire transcriptomes of these samples showed that bulk MEF cultures exposed to doxycycline and AGi were more similar to established iPSCs, whereas MEFs receiving only doxycycline for the same period were more similar to the starting MEF population (Fig. 5a,b). Notably, reprogrammable MEFs exposed to doxycycline and AGi for only 48 hours were already distinguishable from MEFs exposed to doxycycline alone (Supplementary Fig. 7a). MEF-related transcripts (e.g. Col5a, Fbln5, miR-143) were downregulated more rapidly in AGi-treated cells while key pluripotency transcripts including Nanog, Sox2, Zfp42, Zfp296, and miR-290-295 were upregulated much earlier in AGi-exposed cells than in control cells treated with doxycycline alone (Fig. 5d). Moreover, 48 hours of OKSM expression elicited more rapid downregulation of genes associated with FGF signaling, which reportedly blocks iPSC formation29,30 when AGi was present in the culture (Supplementary Fig. 7b). Accordingly, functional annotation analysis of downregulated genes in AGi-treated compared to untreated cells showed enrichment for categories related to differentiation and expression of somatic genes from different lineages (Fig. 5c).


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)

Effect of AGi on gene expression patterns in reprogramming intermediates. (a) Unsupervised clustering of global gene expression analysis of indicated samples. “4–12 days OKSM” represent reprogrammable MEFs exposed for 4–12 days to doxycycline in the absence or presence of AGi. (b) A heat map showing expression levels for genes that were greater than three-fold higher in ESCs relative to MEFs. (c) DAVID functional analysis of genes whose expression is downregulated at least 1.5 fold in response to AGi treatment in MEFs expressing OKSM/OKSM+AGi. Note the prevalence of developmental regulators. A Benjamini-Hochberg (BH) adjusted p-value is presented. (d) Examples of somatic, pluripotency, microRNA, and transient developmental genes that change expression in response to doxycycline/AGi treatment. (e) Expression patterns of gene sets associated with a refractory phenotype (left panel) or transient upregulation during reprogramming (right panel) were compared with expression patterns obtained after OKSM expression in the presence or absence of AGi. Statistical significance is indicated with asterisks (One-tailed Fischer test).
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Related In: Results  -  Collection

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Figure 5: Effect of AGi on gene expression patterns in reprogramming intermediates. (a) Unsupervised clustering of global gene expression analysis of indicated samples. “4–12 days OKSM” represent reprogrammable MEFs exposed for 4–12 days to doxycycline in the absence or presence of AGi. (b) A heat map showing expression levels for genes that were greater than three-fold higher in ESCs relative to MEFs. (c) DAVID functional analysis of genes whose expression is downregulated at least 1.5 fold in response to AGi treatment in MEFs expressing OKSM/OKSM+AGi. Note the prevalence of developmental regulators. A Benjamini-Hochberg (BH) adjusted p-value is presented. (d) Examples of somatic, pluripotency, microRNA, and transient developmental genes that change expression in response to doxycycline/AGi treatment. (e) Expression patterns of gene sets associated with a refractory phenotype (left panel) or transient upregulation during reprogramming (right panel) were compared with expression patterns obtained after OKSM expression in the presence or absence of AGi. Statistical significance is indicated with asterisks (One-tailed Fischer test).
Mentions: Successful reprogramming requires extinction of the somatic program and activation of key pluripotency factors7. Furthermore, cell populations expressing OKSM were reported to transiently upregulate developmental genes10,28; however, the relevance of these changes remains unclear. In an attempt to dissect the molecular consequences of AGi treatment, we performed expression profiling of reprogrammable MEFs exposed to doxycycline for 4, 6, 8, 10, 12 days in the presence or absence of AGi. Unsupervised clustering of the entire transcriptomes of these samples showed that bulk MEF cultures exposed to doxycycline and AGi were more similar to established iPSCs, whereas MEFs receiving only doxycycline for the same period were more similar to the starting MEF population (Fig. 5a,b). Notably, reprogrammable MEFs exposed to doxycycline and AGi for only 48 hours were already distinguishable from MEFs exposed to doxycycline alone (Supplementary Fig. 7a). MEF-related transcripts (e.g. Col5a, Fbln5, miR-143) were downregulated more rapidly in AGi-treated cells while key pluripotency transcripts including Nanog, Sox2, Zfp42, Zfp296, and miR-290-295 were upregulated much earlier in AGi-exposed cells than in control cells treated with doxycycline alone (Fig. 5d). Moreover, 48 hours of OKSM expression elicited more rapid downregulation of genes associated with FGF signaling, which reportedly blocks iPSC formation29,30 when AGi was present in the culture (Supplementary Fig. 7b). Accordingly, functional annotation analysis of downregulated genes in AGi-treated compared to untreated cells showed enrichment for categories related to differentiation and expression of somatic genes from different lineages (Fig. 5c).

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