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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 rescues the reprogramming defect of refractory cells. (a) FACS analysis of reprogramming intermediates in bulk MEF populations at days 5, 7, 9, 11 of OKSM or OKSM+AGi expression. Note the more homogeneous shift of intermediates from THY1+ to THY1-/EPCAM+ cells. (b) Graph summarizing the percentages of EPCAM+ cells depicted in (a). (c) FACS analysis for OCT4-GFP expression in bulk MEF populations at days 5, 7, 9, 11, and 13 of OKSM or OKSM+AGi expression. PE-Cy7 was used as an autofluorescent control with emission filter 750 nm. (d) Graph summarizing the percentages of OCT4-GFP+ cells shown in (c). (e) Schematic outlining the attempt to restore reprogramming potential in THY1+ refractory cells sorted at day 5 of OKSM expression. (f) AP staining for refractory, THY1+ intermediates following continued OKSM expression (doxycycline exposure) under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (g) Quantification for the results shown in (f). Replicate analyses and individual treatment with ascorbic acid and GSK3-beta inhibitor are shown in Supplementary Figure 6. (h) AP staining for cells expressing OKS under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (i) Quantification for the results shown in (h)(n=3 technical replicates).
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Figure 4: AGi rescues the reprogramming defect of refractory cells. (a) FACS analysis of reprogramming intermediates in bulk MEF populations at days 5, 7, 9, 11 of OKSM or OKSM+AGi expression. Note the more homogeneous shift of intermediates from THY1+ to THY1-/EPCAM+ cells. (b) Graph summarizing the percentages of EPCAM+ cells depicted in (a). (c) FACS analysis for OCT4-GFP expression in bulk MEF populations at days 5, 7, 9, 11, and 13 of OKSM or OKSM+AGi expression. PE-Cy7 was used as an autofluorescent control with emission filter 750 nm. (d) Graph summarizing the percentages of OCT4-GFP+ cells shown in (c). (e) Schematic outlining the attempt to restore reprogramming potential in THY1+ refractory cells sorted at day 5 of OKSM expression. (f) AP staining for refractory, THY1+ intermediates following continued OKSM expression (doxycycline exposure) under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (g) Quantification for the results shown in (f). Replicate analyses and individual treatment with ascorbic acid and GSK3-beta inhibitor are shown in Supplementary Figure 6. (h) AP staining for cells expressing OKS under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (i) Quantification for the results shown in (h)(n=3 technical replicates).

Mentions: To gain mechanistic insights into how AGi treatment may influence the reprogramming process, we analyzed intermediate time points during iPSC formation by FACS using previously described surface markers and a reporter allele10,11. MEFs undergoing successful reprogramming initially downregulate the fibroblast marker THY1, followed by successive activation of the intermediate marker EPCAM, and subsequent expression of OCT4-GFP. In agreement with previous studies, we detected downregulation of THY1 in two thirds of the population and only a small fraction of intermediates expressing either EPCAM (22%) or OCT4-GFP (0.8%) by day 11 of reprogramming (Fig. 4a–d). In contrast, exposure of cells to AGi for the same duration triggered these phenotypic changes in a much larger fraction of cells (92% THY1−, 87% EPCAM+, 48% OCT4-GFP+) (Fig. 4a–d). These results thus suggested that AGi supplementation reduces a high fraction of OKSM-expressing cells that typically fail to form iPSCs. To directly test this hypothesis, we induced OKSM expression in MEFs for 5 days and sorted intermediates based on THY1 positivity and SSEA1 negativity, which identify cells that have become refractory to iPSC induction after day 3 (Fig. 4e)10,11. While THY1+ cells exposed to regular culture conditions failed to yield iPSCs, addition of AGi restored their potential to produce doxycycline-independent iPSCs (Fig. 4f,g, Supplementary Fig. 6). Thus, AGi’s effect on reprogramming is at least in part explained by its ability to prevent cells from arresting at intermediate stages of iPSC induction.


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 rescues the reprogramming defect of refractory cells. (a) FACS analysis of reprogramming intermediates in bulk MEF populations at days 5, 7, 9, 11 of OKSM or OKSM+AGi expression. Note the more homogeneous shift of intermediates from THY1+ to THY1-/EPCAM+ cells. (b) Graph summarizing the percentages of EPCAM+ cells depicted in (a). (c) FACS analysis for OCT4-GFP expression in bulk MEF populations at days 5, 7, 9, 11, and 13 of OKSM or OKSM+AGi expression. PE-Cy7 was used as an autofluorescent control with emission filter 750 nm. (d) Graph summarizing the percentages of OCT4-GFP+ cells shown in (c). (e) Schematic outlining the attempt to restore reprogramming potential in THY1+ refractory cells sorted at day 5 of OKSM expression. (f) AP staining for refractory, THY1+ intermediates following continued OKSM expression (doxycycline exposure) under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (g) Quantification for the results shown in (f). Replicate analyses and individual treatment with ascorbic acid and GSK3-beta inhibitor are shown in Supplementary Figure 6. (h) AP staining for cells expressing OKS under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (i) Quantification for the results shown in (h)(n=3 technical replicates).
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Figure 4: AGi rescues the reprogramming defect of refractory cells. (a) FACS analysis of reprogramming intermediates in bulk MEF populations at days 5, 7, 9, 11 of OKSM or OKSM+AGi expression. Note the more homogeneous shift of intermediates from THY1+ to THY1-/EPCAM+ cells. (b) Graph summarizing the percentages of EPCAM+ cells depicted in (a). (c) FACS analysis for OCT4-GFP expression in bulk MEF populations at days 5, 7, 9, 11, and 13 of OKSM or OKSM+AGi expression. PE-Cy7 was used as an autofluorescent control with emission filter 750 nm. (d) Graph summarizing the percentages of OCT4-GFP+ cells shown in (c). (e) Schematic outlining the attempt to restore reprogramming potential in THY1+ refractory cells sorted at day 5 of OKSM expression. (f) AP staining for refractory, THY1+ intermediates following continued OKSM expression (doxycycline exposure) under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (g) Quantification for the results shown in (f). Replicate analyses and individual treatment with ascorbic acid and GSK3-beta inhibitor are shown in Supplementary Figure 6. (h) AP staining for cells expressing OKS under the indicated conditions. Doxycylcine was removed for an additional 4 days prior to analysis to ensure transgene independence. (i) Quantification for the results shown in (h)(n=3 technical replicates).
Mentions: To gain mechanistic insights into how AGi treatment may influence the reprogramming process, we analyzed intermediate time points during iPSC formation by FACS using previously described surface markers and a reporter allele10,11. MEFs undergoing successful reprogramming initially downregulate the fibroblast marker THY1, followed by successive activation of the intermediate marker EPCAM, and subsequent expression of OCT4-GFP. In agreement with previous studies, we detected downregulation of THY1 in two thirds of the population and only a small fraction of intermediates expressing either EPCAM (22%) or OCT4-GFP (0.8%) by day 11 of reprogramming (Fig. 4a–d). In contrast, exposure of cells to AGi for the same duration triggered these phenotypic changes in a much larger fraction of cells (92% THY1−, 87% EPCAM+, 48% OCT4-GFP+) (Fig. 4a–d). These results thus suggested that AGi supplementation reduces a high fraction of OKSM-expressing cells that typically fail to form iPSCs. To directly test this hypothesis, we induced OKSM expression in MEFs for 5 days and sorted intermediates based on THY1 positivity and SSEA1 negativity, which identify cells that have become refractory to iPSC induction after day 3 (Fig. 4e)10,11. While THY1+ cells exposed to regular culture conditions failed to yield iPSCs, addition of AGi restored their potential to produce doxycycline-independent iPSCs (Fig. 4f,g, Supplementary Fig. 6). Thus, AGi’s effect on reprogramming is at least in part explained by its ability to prevent cells from arresting at intermediate stages of iPSC induction.

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