Limits...
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.

Show MeSH

Related in: MedlinePlus

Clonal analysis of GMP reprogramming indicates synchronous reprogramming with AGi. (a) Schematic for clonal reprogramming assay in 96-well plates. (b) Representative example from three technical replicates of clonal reprogramming analysis of GMPs at day 6 of OKSM expression in control (doxycycline alone) vs. AGi (doxycycline plus AGi) setting. Green fields represent wells of a 96 well-plate that contained 53% or more OCT4-GFP+ cells and were thus regarded iPSCs (see also text and methods section); gray fields were GFP-negative or contained less than 53% OCT4-GFP+ cells; black fields represent wells in which fewer than 10 cells were detected. (c) Time course analysis of clonal iPSC formation in the presence or absence of AGi. (d) Examination of intra-well heterogeneity of OCT4-GFP expression in GMPs during the course of reprogramming. Box and whisker plots were used in which the band in the middle of the box represents the median; the top and bottom part of the box represent the first and third quartiles. The whiskers represent the lowest and highest value within 1.5-fold of the inter-quartile ranges.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4326224&req=5

Figure 3: Clonal analysis of GMP reprogramming indicates synchronous reprogramming with AGi. (a) Schematic for clonal reprogramming assay in 96-well plates. (b) Representative example from three technical replicates of clonal reprogramming analysis of GMPs at day 6 of OKSM expression in control (doxycycline alone) vs. AGi (doxycycline plus AGi) setting. Green fields represent wells of a 96 well-plate that contained 53% or more OCT4-GFP+ cells and were thus regarded iPSCs (see also text and methods section); gray fields were GFP-negative or contained less than 53% OCT4-GFP+ cells; black fields represent wells in which fewer than 10 cells were detected. (c) Time course analysis of clonal iPSC formation in the presence or absence of AGi. (d) Examination of intra-well heterogeneity of OCT4-GFP expression in GMPs during the course of reprogramming. Box and whisker plots were used in which the band in the middle of the box represents the median; the top and bottom part of the box represent the first and third quartiles. The whiskers represent the lowest and highest value within 1.5-fold of the inter-quartile ranges.

Mentions: To accurately quantify reprogramming efficiency and kinetics, we sorted single blood progenitor cells carrying the tetOP-OKSM/tetOP-OKSmC alleles as well as an Oct4-GFP knock-in reporter15 by fluorescence-activated cell sorting (FACS) into individual wells of 96-well plates (Fig. 3a). We then evaluated OCT4-GFP activation at discrete time points (Fig. 3b,c). For GMPs, wells containing 53% or more OCT4-GFP+ cells at any given time point were considered positive hits for reprogrammed iPSCs. We determined this cutoff empirically to eliminate false-positive “iPSC calls” that were prevalent at earlier time points due to low cell numbers and autofluorescent feeders (see methods section). By applying these stringent criteria, we noticed a marked difference in reprogramming efficiency with and without AGi at day 6 of induction (Fig. 3b). Examination of reprogramming efficiencies for cells treated with doxycycline alone revealed that roughly 20% of GMPs became OCT4-GFP+ by day 8, ~50% by day 20 and close to 100% by day 30 (Fig. 3c). These reprogramming efficiencies were two-fold higher than previously observed by our lab when comparing similar time points (25% at day 15)5, which is likely due to the superior reprogramming system used here15. Remarkably, exposure of replicate plates to AGi yielded over 20% OCT4-GFP+ clones by day 3, 80% by day 4 and over 95% by day 5 (Fig. 3c). These results indicate that AGi treatment facilitates more synchronous iPSC formation from myeloid progenitors when using a clonal reprogramming assay.


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)

Clonal analysis of GMP reprogramming indicates synchronous reprogramming with AGi. (a) Schematic for clonal reprogramming assay in 96-well plates. (b) Representative example from three technical replicates of clonal reprogramming analysis of GMPs at day 6 of OKSM expression in control (doxycycline alone) vs. AGi (doxycycline plus AGi) setting. Green fields represent wells of a 96 well-plate that contained 53% or more OCT4-GFP+ cells and were thus regarded iPSCs (see also text and methods section); gray fields were GFP-negative or contained less than 53% OCT4-GFP+ cells; black fields represent wells in which fewer than 10 cells were detected. (c) Time course analysis of clonal iPSC formation in the presence or absence of AGi. (d) Examination of intra-well heterogeneity of OCT4-GFP expression in GMPs during the course of reprogramming. Box and whisker plots were used in which the band in the middle of the box represents the median; the top and bottom part of the box represent the first and third quartiles. The whiskers represent the lowest and highest value within 1.5-fold of the inter-quartile ranges.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Clonal analysis of GMP reprogramming indicates synchronous reprogramming with AGi. (a) Schematic for clonal reprogramming assay in 96-well plates. (b) Representative example from three technical replicates of clonal reprogramming analysis of GMPs at day 6 of OKSM expression in control (doxycycline alone) vs. AGi (doxycycline plus AGi) setting. Green fields represent wells of a 96 well-plate that contained 53% or more OCT4-GFP+ cells and were thus regarded iPSCs (see also text and methods section); gray fields were GFP-negative or contained less than 53% OCT4-GFP+ cells; black fields represent wells in which fewer than 10 cells were detected. (c) Time course analysis of clonal iPSC formation in the presence or absence of AGi. (d) Examination of intra-well heterogeneity of OCT4-GFP expression in GMPs during the course of reprogramming. Box and whisker plots were used in which the band in the middle of the box represents the median; the top and bottom part of the box represent the first and third quartiles. The whiskers represent the lowest and highest value within 1.5-fold of the inter-quartile ranges.
Mentions: To accurately quantify reprogramming efficiency and kinetics, we sorted single blood progenitor cells carrying the tetOP-OKSM/tetOP-OKSmC alleles as well as an Oct4-GFP knock-in reporter15 by fluorescence-activated cell sorting (FACS) into individual wells of 96-well plates (Fig. 3a). We then evaluated OCT4-GFP activation at discrete time points (Fig. 3b,c). For GMPs, wells containing 53% or more OCT4-GFP+ cells at any given time point were considered positive hits for reprogrammed iPSCs. We determined this cutoff empirically to eliminate false-positive “iPSC calls” that were prevalent at earlier time points due to low cell numbers and autofluorescent feeders (see methods section). By applying these stringent criteria, we noticed a marked difference in reprogramming efficiency with and without AGi at day 6 of induction (Fig. 3b). Examination of reprogramming efficiencies for cells treated with doxycycline alone revealed that roughly 20% of GMPs became OCT4-GFP+ by day 8, ~50% by day 20 and close to 100% by day 30 (Fig. 3c). These reprogramming efficiencies were two-fold higher than previously observed by our lab when comparing similar time points (25% at day 15)5, which is likely due to the superior reprogramming system used here15. Remarkably, exposure of replicate plates to AGi yielded over 20% OCT4-GFP+ clones by day 3, 80% by day 4 and over 95% by day 5 (Fig. 3c). These results indicate that AGi treatment facilitates more synchronous iPSC formation from myeloid progenitors when using a clonal reprogramming assay.

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