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KLF4 N-terminal variance modulates induced reprogramming to pluripotency.

Kim SI, Oceguera-Yanez F, Hirohata R, Linker S, Okita K, Yamada Y, Yamamoto T, Yamanaka S, Woltjen K - Stem Cell Reports (2015)

Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.

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KLF4 Stoichiometry Affects Reprogramming Phenotypes(A) Reprogramming with polycistronic OMS + monocistronic Klf4S or Klf4L leads to nearly identical phenotypes, as analyzed by FACS for mCherry or SSEA-1 on d8 (left) and mCherry versus Nanog-GFP on d18 (right).(B) Western blot analysis of OCT3/4, SOX2, c-MYC, and KLF4 in OSKM or OKMS transfected MEFs cultured for 2 days with or without dox treatment. Actin was used as a loading control. Arrows show the two KLF4 isoforms. Note that SOX2 and c-MYC also differ in size based on their positions relative to 2A peptides in the polycistronic cassette (Figure 1D). Uncropped data are provided in Figure S2G.(C–F) Supplementation of PB-TAC-OKMS was performed by co-transfection of additional factors in PB-TAB transposons. β-geo, lacZ-neo fusion gene.(D) AP staining on d10 after transfection with OKMS alone or in combination with Oct3/4, Sox2, c-Myc, Klf4S, or Klf4L. Scale bar, 4,000 μm.(E) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+. Scale bars, 4,000 μm (full well) and 1,000 μm (inset).(F) FACS analysis of mCherry and Nanog-GFP fractions in the d18 SSEA-1+ population of the cultures shown in (E). The results in (D)–(F) are representative of the results of at least three independent experiments (summarized in G and H).(G) Quantification of the effects of factor supplementation on AP+ colony formation (D). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.(H) Percentages of mCherry+ and Nanog-GFP+ populations from FACS analysis of factor supplementation (F). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.See also Figure S2.
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fig2: KLF4 Stoichiometry Affects Reprogramming Phenotypes(A) Reprogramming with polycistronic OMS + monocistronic Klf4S or Klf4L leads to nearly identical phenotypes, as analyzed by FACS for mCherry or SSEA-1 on d8 (left) and mCherry versus Nanog-GFP on d18 (right).(B) Western blot analysis of OCT3/4, SOX2, c-MYC, and KLF4 in OSKM or OKMS transfected MEFs cultured for 2 days with or without dox treatment. Actin was used as a loading control. Arrows show the two KLF4 isoforms. Note that SOX2 and c-MYC also differ in size based on their positions relative to 2A peptides in the polycistronic cassette (Figure 1D). Uncropped data are provided in Figure S2G.(C–F) Supplementation of PB-TAC-OKMS was performed by co-transfection of additional factors in PB-TAB transposons. β-geo, lacZ-neo fusion gene.(D) AP staining on d10 after transfection with OKMS alone or in combination with Oct3/4, Sox2, c-Myc, Klf4S, or Klf4L. Scale bar, 4,000 μm.(E) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+. Scale bars, 4,000 μm (full well) and 1,000 μm (inset).(F) FACS analysis of mCherry and Nanog-GFP fractions in the d18 SSEA-1+ population of the cultures shown in (E). The results in (D)–(F) are representative of the results of at least three independent experiments (summarized in G and H).(G) Quantification of the effects of factor supplementation on AP+ colony formation (D). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.(H) Percentages of mCherry+ and Nanog-GFP+ populations from FACS analysis of factor supplementation (F). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.See also Figure S2.

Mentions: Considering the remarkable reprogramming phenotypes observed with polycistronic cassettes, we next addressed whether monocistronic expression of Klf4S or Klf4L in combination with polycistronic Oct3/4, Sox2, and c-Myc (OMS) had any detectable effect on iPSC derivation. OMS alone did not foster expansion of mCherry+ cells, which remained at a steady ∼6% through d18, nor did it activate SSEA-1 or Nanog-GFP expression (data not shown). Interestingly, expression of either Klf4 variant in combination with OMS resulted in nearly indistinguishable reprogramming phenotypes using the assays outlined in Figure 1 (see also Figures 2A, S2D, and S2E). These data demonstrate that the phenotypes associated with KLF4S and KLF4L arise mainly in the context of polycistronic factor linkage.


KLF4 N-terminal variance modulates induced reprogramming to pluripotency.

Kim SI, Oceguera-Yanez F, Hirohata R, Linker S, Okita K, Yamada Y, Yamamoto T, Yamanaka S, Woltjen K - Stem Cell Reports (2015)

KLF4 Stoichiometry Affects Reprogramming Phenotypes(A) Reprogramming with polycistronic OMS + monocistronic Klf4S or Klf4L leads to nearly identical phenotypes, as analyzed by FACS for mCherry or SSEA-1 on d8 (left) and mCherry versus Nanog-GFP on d18 (right).(B) Western blot analysis of OCT3/4, SOX2, c-MYC, and KLF4 in OSKM or OKMS transfected MEFs cultured for 2 days with or without dox treatment. Actin was used as a loading control. Arrows show the two KLF4 isoforms. Note that SOX2 and c-MYC also differ in size based on their positions relative to 2A peptides in the polycistronic cassette (Figure 1D). Uncropped data are provided in Figure S2G.(C–F) Supplementation of PB-TAC-OKMS was performed by co-transfection of additional factors in PB-TAB transposons. β-geo, lacZ-neo fusion gene.(D) AP staining on d10 after transfection with OKMS alone or in combination with Oct3/4, Sox2, c-Myc, Klf4S, or Klf4L. Scale bar, 4,000 μm.(E) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+. Scale bars, 4,000 μm (full well) and 1,000 μm (inset).(F) FACS analysis of mCherry and Nanog-GFP fractions in the d18 SSEA-1+ population of the cultures shown in (E). The results in (D)–(F) are representative of the results of at least three independent experiments (summarized in G and H).(G) Quantification of the effects of factor supplementation on AP+ colony formation (D). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.(H) Percentages of mCherry+ and Nanog-GFP+ populations from FACS analysis of factor supplementation (F). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.See also Figure S2.
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fig2: KLF4 Stoichiometry Affects Reprogramming Phenotypes(A) Reprogramming with polycistronic OMS + monocistronic Klf4S or Klf4L leads to nearly identical phenotypes, as analyzed by FACS for mCherry or SSEA-1 on d8 (left) and mCherry versus Nanog-GFP on d18 (right).(B) Western blot analysis of OCT3/4, SOX2, c-MYC, and KLF4 in OSKM or OKMS transfected MEFs cultured for 2 days with or without dox treatment. Actin was used as a loading control. Arrows show the two KLF4 isoforms. Note that SOX2 and c-MYC also differ in size based on their positions relative to 2A peptides in the polycistronic cassette (Figure 1D). Uncropped data are provided in Figure S2G.(C–F) Supplementation of PB-TAC-OKMS was performed by co-transfection of additional factors in PB-TAB transposons. β-geo, lacZ-neo fusion gene.(D) AP staining on d10 after transfection with OKMS alone or in combination with Oct3/4, Sox2, c-Myc, Klf4S, or Klf4L. Scale bar, 4,000 μm.(E) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+. Scale bars, 4,000 μm (full well) and 1,000 μm (inset).(F) FACS analysis of mCherry and Nanog-GFP fractions in the d18 SSEA-1+ population of the cultures shown in (E). The results in (D)–(F) are representative of the results of at least three independent experiments (summarized in G and H).(G) Quantification of the effects of factor supplementation on AP+ colony formation (D). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.(H) Percentages of mCherry+ and Nanog-GFP+ populations from FACS analysis of factor supplementation (F). Means ± SE for three independent experiments. n.s., not significant. ∗p < 0.05, Student’s t test.See also Figure S2.
Mentions: Considering the remarkable reprogramming phenotypes observed with polycistronic cassettes, we next addressed whether monocistronic expression of Klf4S or Klf4L in combination with polycistronic Oct3/4, Sox2, and c-Myc (OMS) had any detectable effect on iPSC derivation. OMS alone did not foster expansion of mCherry+ cells, which remained at a steady ∼6% through d18, nor did it activate SSEA-1 or Nanog-GFP expression (data not shown). Interestingly, expression of either Klf4 variant in combination with OMS resulted in nearly indistinguishable reprogramming phenotypes using the assays outlined in Figure 1 (see also Figures 2A, S2D, and S2E). These data demonstrate that the phenotypes associated with KLF4S and KLF4L arise mainly in the context of polycistronic factor linkage.

Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.

Show MeSH
Related in: MedlinePlus