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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 Isoforms Underlie Phenotypic Differences in Reprogramming(A) Diagram of the PB reprogramming system and analysis steps. The PB-TAC transposon delivers inducible, reporter-linked (mCherry), polycistronic reprogramming cassettes (Poly) into ROSA-rtTA; Nanog-GFP MEFs (d-1). Cultures are induced with dox (d0, dox; filled arrows) and harvested on d8 for FACS and passage for late-stage analysis (d18). For AP staining, cultures are maintained without passage until d10. Full reprogramming and transgene independence is assessed by dox withdrawal from d18 to d24 (open arrows). Predicted expression periods for the mCherry and GFP reporters during iPSC derivation are shown with red and green gradated bars. Blue polygons represent PB 3′ (left) and 5′ (right) inverted terminal repeats. tetO, dox-responsive promoter; IRES, internal ribosome entry signal; pA, polyadenylation signal.(B) AP staining on d10 of OSKM and OKMS reprogramming cultures. Scale bar, 4,000 μm.(C) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+ (left). Scale bars, 4,000 μm (full well) and 1,000 μm (inset). FACS analysis of mCherry and Nanog-GFP expression in d18 Total and SSEA-1+ populations (right).(D) Polycistronic cassette structure and sequence of the 2A-Klf4 N-terminal cleavage junctions. Cloned cDNA is compared with murine Klf4 GenBank mRNA sequences (U20344.1 and U70662.1), indicating the position of the predicted initiation codons and amino acid translations. The N-terminal 9aa of U20344.1 were introduced into OKMS (Klf4S) to produce OK+9MS (Klf4L).(E and F) The OK+9MS construct was evaluated according to the assays outlined in (A)–(C). The results in (B), (C), (E), and (F) are representative of the results from at least three independent experiments (summarized in Figures S1A and S1B).(G and H) Time-course analysis of mCherry+ cell expansion (G) and SSEA-1+ fractions (H) from OSKM, OKMS, and OK+9MS transfections cultured for the indicated number of days before (2, 4, 6, 8) and after (10, 14, and 18) passage. Means ± SE for three independent experiments.(I and J) Time-course analysis of mCherry silencing (left) and Nanog-GFP activation (right) in SSEA-1+ cells on the indicated number of days after day 8 passage (10, 14, and 18). Means ± SE for three independent experiments.See also Figure S1.
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fig1: KLF4 Isoforms Underlie Phenotypic Differences in Reprogramming(A) Diagram of the PB reprogramming system and analysis steps. The PB-TAC transposon delivers inducible, reporter-linked (mCherry), polycistronic reprogramming cassettes (Poly) into ROSA-rtTA; Nanog-GFP MEFs (d-1). Cultures are induced with dox (d0, dox; filled arrows) and harvested on d8 for FACS and passage for late-stage analysis (d18). For AP staining, cultures are maintained without passage until d10. Full reprogramming and transgene independence is assessed by dox withdrawal from d18 to d24 (open arrows). Predicted expression periods for the mCherry and GFP reporters during iPSC derivation are shown with red and green gradated bars. Blue polygons represent PB 3′ (left) and 5′ (right) inverted terminal repeats. tetO, dox-responsive promoter; IRES, internal ribosome entry signal; pA, polyadenylation signal.(B) AP staining on d10 of OSKM and OKMS reprogramming cultures. Scale bar, 4,000 μm.(C) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+ (left). Scale bars, 4,000 μm (full well) and 1,000 μm (inset). FACS analysis of mCherry and Nanog-GFP expression in d18 Total and SSEA-1+ populations (right).(D) Polycistronic cassette structure and sequence of the 2A-Klf4 N-terminal cleavage junctions. Cloned cDNA is compared with murine Klf4 GenBank mRNA sequences (U20344.1 and U70662.1), indicating the position of the predicted initiation codons and amino acid translations. The N-terminal 9aa of U20344.1 were introduced into OKMS (Klf4S) to produce OK+9MS (Klf4L).(E and F) The OK+9MS construct was evaluated according to the assays outlined in (A)–(C). The results in (B), (C), (E), and (F) are representative of the results from at least three independent experiments (summarized in Figures S1A and S1B).(G and H) Time-course analysis of mCherry+ cell expansion (G) and SSEA-1+ fractions (H) from OSKM, OKMS, and OK+9MS transfections cultured for the indicated number of days before (2, 4, 6, 8) and after (10, 14, and 18) passage. Means ± SE for three independent experiments.(I and J) Time-course analysis of mCherry silencing (left) and Nanog-GFP activation (right) in SSEA-1+ cells on the indicated number of days after day 8 passage (10, 14, and 18). Means ± SE for three independent experiments.See also Figure S1.

Mentions: Reprogramming studies in mouse have made use of unique polycistronic factor arrangements and delivery vectors. For uniform evaluation of factor-order effects, we employed a fundamental reprogramming scheme based on factor transposition in MEFs (Woltjen et al., 2009). The PB transposon vector (PB-TAC) employs doxycycline (dox)-responsive reprogramming cassette expression co-incidentally with a mCherry reporter (Figure 1A). ROSA-rtTA; Nanog-GFP MEFs combine the m2-rtTA transactivator (Ohnishi et al., 2014) with a Nanog-GFP reporter (Okita et al., 2007). Thus, dox-responsive, PB-TAC-transgenic cells can be monitored throughout reprogramming initiation and maturation (day 2 [d2]–14) by mCherry fluorescence, while stabilization of pluripotency (d14–18) is indicated by activation of Nanog-GFP. Gain of factor independence through autonomous transgene silencing despite continued dox treatment is an established hallmark of the stabilization phase (Golipour et al., 2012), signaled here by a decrease in mCherry expression. For all polycistronic reprogramming cassettes tested, we routinely passaged populations on d8 and d18 using equal cell numbers without fractionation for extended culture and fluorescence-activated cell sorting (FACS) analysis. Dox-independent maintenance of iPSCs was verified by culture until d24, after which pluripotency was assayed by gene expression array and chimera contribution.


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 Isoforms Underlie Phenotypic Differences in Reprogramming(A) Diagram of the PB reprogramming system and analysis steps. The PB-TAC transposon delivers inducible, reporter-linked (mCherry), polycistronic reprogramming cassettes (Poly) into ROSA-rtTA; Nanog-GFP MEFs (d-1). Cultures are induced with dox (d0, dox; filled arrows) and harvested on d8 for FACS and passage for late-stage analysis (d18). For AP staining, cultures are maintained without passage until d10. Full reprogramming and transgene independence is assessed by dox withdrawal from d18 to d24 (open arrows). Predicted expression periods for the mCherry and GFP reporters during iPSC derivation are shown with red and green gradated bars. Blue polygons represent PB 3′ (left) and 5′ (right) inverted terminal repeats. tetO, dox-responsive promoter; IRES, internal ribosome entry signal; pA, polyadenylation signal.(B) AP staining on d10 of OSKM and OKMS reprogramming cultures. Scale bar, 4,000 μm.(C) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+ (left). Scale bars, 4,000 μm (full well) and 1,000 μm (inset). FACS analysis of mCherry and Nanog-GFP expression in d18 Total and SSEA-1+ populations (right).(D) Polycistronic cassette structure and sequence of the 2A-Klf4 N-terminal cleavage junctions. Cloned cDNA is compared with murine Klf4 GenBank mRNA sequences (U20344.1 and U70662.1), indicating the position of the predicted initiation codons and amino acid translations. The N-terminal 9aa of U20344.1 were introduced into OKMS (Klf4S) to produce OK+9MS (Klf4L).(E and F) The OK+9MS construct was evaluated according to the assays outlined in (A)–(C). The results in (B), (C), (E), and (F) are representative of the results from at least three independent experiments (summarized in Figures S1A and S1B).(G and H) Time-course analysis of mCherry+ cell expansion (G) and SSEA-1+ fractions (H) from OSKM, OKMS, and OK+9MS transfections cultured for the indicated number of days before (2, 4, 6, 8) and after (10, 14, and 18) passage. Means ± SE for three independent experiments.(I and J) Time-course analysis of mCherry silencing (left) and Nanog-GFP activation (right) in SSEA-1+ cells on the indicated number of days after day 8 passage (10, 14, and 18). Means ± SE for three independent experiments.See also Figure S1.
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fig1: KLF4 Isoforms Underlie Phenotypic Differences in Reprogramming(A) Diagram of the PB reprogramming system and analysis steps. The PB-TAC transposon delivers inducible, reporter-linked (mCherry), polycistronic reprogramming cassettes (Poly) into ROSA-rtTA; Nanog-GFP MEFs (d-1). Cultures are induced with dox (d0, dox; filled arrows) and harvested on d8 for FACS and passage for late-stage analysis (d18). For AP staining, cultures are maintained without passage until d10. Full reprogramming and transgene independence is assessed by dox withdrawal from d18 to d24 (open arrows). Predicted expression periods for the mCherry and GFP reporters during iPSC derivation are shown with red and green gradated bars. Blue polygons represent PB 3′ (left) and 5′ (right) inverted terminal repeats. tetO, dox-responsive promoter; IRES, internal ribosome entry signal; pA, polyadenylation signal.(B) AP staining on d10 of OSKM and OKMS reprogramming cultures. Scale bar, 4,000 μm.(C) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry+ and Nanog-GFP+ (left). Scale bars, 4,000 μm (full well) and 1,000 μm (inset). FACS analysis of mCherry and Nanog-GFP expression in d18 Total and SSEA-1+ populations (right).(D) Polycistronic cassette structure and sequence of the 2A-Klf4 N-terminal cleavage junctions. Cloned cDNA is compared with murine Klf4 GenBank mRNA sequences (U20344.1 and U70662.1), indicating the position of the predicted initiation codons and amino acid translations. The N-terminal 9aa of U20344.1 were introduced into OKMS (Klf4S) to produce OK+9MS (Klf4L).(E and F) The OK+9MS construct was evaluated according to the assays outlined in (A)–(C). The results in (B), (C), (E), and (F) are representative of the results from at least three independent experiments (summarized in Figures S1A and S1B).(G and H) Time-course analysis of mCherry+ cell expansion (G) and SSEA-1+ fractions (H) from OSKM, OKMS, and OK+9MS transfections cultured for the indicated number of days before (2, 4, 6, 8) and after (10, 14, and 18) passage. Means ± SE for three independent experiments.(I and J) Time-course analysis of mCherry silencing (left) and Nanog-GFP activation (right) in SSEA-1+ cells on the indicated number of days after day 8 passage (10, 14, and 18). Means ± SE for three independent experiments.See also Figure S1.
Mentions: Reprogramming studies in mouse have made use of unique polycistronic factor arrangements and delivery vectors. For uniform evaluation of factor-order effects, we employed a fundamental reprogramming scheme based on factor transposition in MEFs (Woltjen et al., 2009). The PB transposon vector (PB-TAC) employs doxycycline (dox)-responsive reprogramming cassette expression co-incidentally with a mCherry reporter (Figure 1A). ROSA-rtTA; Nanog-GFP MEFs combine the m2-rtTA transactivator (Ohnishi et al., 2014) with a Nanog-GFP reporter (Okita et al., 2007). Thus, dox-responsive, PB-TAC-transgenic cells can be monitored throughout reprogramming initiation and maturation (day 2 [d2]–14) by mCherry fluorescence, while stabilization of pluripotency (d14–18) is indicated by activation of Nanog-GFP. Gain of factor independence through autonomous transgene silencing despite continued dox treatment is an established hallmark of the stabilization phase (Golipour et al., 2012), signaled here by a decrease in mCherry expression. For all polycistronic reprogramming cassettes tested, we routinely passaged populations on d8 and d18 using equal cell numbers without fractionation for extended culture and fluorescence-activated cell sorting (FACS) analysis. Dox-independent maintenance of iPSCs was verified by culture until d24, after which pluripotency was assayed by gene expression array and chimera contribution.

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