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Tbx3 improves the germ-line competency of induced pluripotent stem cells.

Han J, Yuan P, Yang H, Zhang J, Soh BS, Li P, Lim SL, Cao S, Tay J, Orlov YL, Lufkin T, Ng HH, Tam WL, Lim B - Nature (2010)

Bottom Line: Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency.Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1.This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, 138672, Singapore.

ABSTRACT
Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

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Transcriptome of iPS cell clones generated with different combinations of reprogramming factors. (A) Hierarchical clustering of global expression profiling showed that OSKT and OSK clones are more similar to wild-type ESCs than OSE, but indistinguishable from each other based on correlation coefficient. R2 value was obtained from the average individual gene signal intensity of all iPS clones in each combination and compared against R1 ESCs. The independent transduction experiments where the clones were isolated are denoted as A, B, C, and D. (B) Analysis of individual ESC-associated gene profiles revealed a subset that could distinguish OSKT from OSK-derived iPS cells. ‘Distinguishing’ ESC genes were expressed at levels similar between OSKT and ESCs but significantly lower in OSK. The majority of other ESC-associated genes were ‘non-distinguishing’ and present at levels similar between both OSKT and OSK. * denotes significantly different from OSKT; + denotes significantly different from ESC; p<0.05. Changes in gene expression based on microarray were confirmed with qPCR (Figure S11).
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Figure 3: Transcriptome of iPS cell clones generated with different combinations of reprogramming factors. (A) Hierarchical clustering of global expression profiling showed that OSKT and OSK clones are more similar to wild-type ESCs than OSE, but indistinguishable from each other based on correlation coefficient. R2 value was obtained from the average individual gene signal intensity of all iPS clones in each combination and compared against R1 ESCs. The independent transduction experiments where the clones were isolated are denoted as A, B, C, and D. (B) Analysis of individual ESC-associated gene profiles revealed a subset that could distinguish OSKT from OSK-derived iPS cells. ‘Distinguishing’ ESC genes were expressed at levels similar between OSKT and ESCs but significantly lower in OSK. The majority of other ESC-associated genes were ‘non-distinguishing’ and present at levels similar between both OSKT and OSK. * denotes significantly different from OSKT; + denotes significantly different from ESC; p<0.05. Changes in gene expression based on microarray were confirmed with qPCR (Figure S11).

Mentions: Next we sought to examine the differences in global transcriptome profiles between iPS cells generated using different factor combinations. To eliminate variations that could have arisen from handling techniques, and to ensure the reproducibility of properties inherent to iPS cell lines generated with different combinations, clones were isolated from two or more independent transduction experiments. Hierarchical clustering revealed that the recently reported iPS cells generated with OS + Esrrb (OSE)7 were most dissimilar to wild-type R1 and D3 ESCs with a correlation coefficient (R2) of 0.92 (Figure 3A). Both OSK and OSKT iPS cells bore closer resemblance to ESCs but were indistinguishable from each other; R2 = 0.94. Hence, global expression profiling was not sufficiently sensitive in detecting differences between these clones. Closer examination of specific gene level alterations, however, revealed key differences (Figure 3B; Figure S11). We compared the pluripotency-associated gene levels among OSK, OSKT iPS and ES cells. The majority of these genes such as Sall4, Tcf3, Sox2, Zfx, Lin28, Utf1 and Zic3 were non-distinguishing between OSKT and OSK cells as their levels were similar. Surprisingly, a small subset of distinguishing features appeared to define OSKT from OSK cells. Oct4, Nanog, Gdf3, Dppa4 and Tbx3 levels in OSKT cells were equivalent to ESCs, but significantly reduced in OSK cells. This suggests that exogenous Tbx3 may be crucial for assisting in re-establishing proper levels of certain ESC factors critical for the induction of pluripotency that cannot be completely achieved with OSK alone.


Tbx3 improves the germ-line competency of induced pluripotent stem cells.

Han J, Yuan P, Yang H, Zhang J, Soh BS, Li P, Lim SL, Cao S, Tay J, Orlov YL, Lufkin T, Ng HH, Tam WL, Lim B - Nature (2010)

Transcriptome of iPS cell clones generated with different combinations of reprogramming factors. (A) Hierarchical clustering of global expression profiling showed that OSKT and OSK clones are more similar to wild-type ESCs than OSE, but indistinguishable from each other based on correlation coefficient. R2 value was obtained from the average individual gene signal intensity of all iPS clones in each combination and compared against R1 ESCs. The independent transduction experiments where the clones were isolated are denoted as A, B, C, and D. (B) Analysis of individual ESC-associated gene profiles revealed a subset that could distinguish OSKT from OSK-derived iPS cells. ‘Distinguishing’ ESC genes were expressed at levels similar between OSKT and ESCs but significantly lower in OSK. The majority of other ESC-associated genes were ‘non-distinguishing’ and present at levels similar between both OSKT and OSK. * denotes significantly different from OSKT; + denotes significantly different from ESC; p<0.05. Changes in gene expression based on microarray were confirmed with qPCR (Figure S11).
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Related In: Results  -  Collection

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Figure 3: Transcriptome of iPS cell clones generated with different combinations of reprogramming factors. (A) Hierarchical clustering of global expression profiling showed that OSKT and OSK clones are more similar to wild-type ESCs than OSE, but indistinguishable from each other based on correlation coefficient. R2 value was obtained from the average individual gene signal intensity of all iPS clones in each combination and compared against R1 ESCs. The independent transduction experiments where the clones were isolated are denoted as A, B, C, and D. (B) Analysis of individual ESC-associated gene profiles revealed a subset that could distinguish OSKT from OSK-derived iPS cells. ‘Distinguishing’ ESC genes were expressed at levels similar between OSKT and ESCs but significantly lower in OSK. The majority of other ESC-associated genes were ‘non-distinguishing’ and present at levels similar between both OSKT and OSK. * denotes significantly different from OSKT; + denotes significantly different from ESC; p<0.05. Changes in gene expression based on microarray were confirmed with qPCR (Figure S11).
Mentions: Next we sought to examine the differences in global transcriptome profiles between iPS cells generated using different factor combinations. To eliminate variations that could have arisen from handling techniques, and to ensure the reproducibility of properties inherent to iPS cell lines generated with different combinations, clones were isolated from two or more independent transduction experiments. Hierarchical clustering revealed that the recently reported iPS cells generated with OS + Esrrb (OSE)7 were most dissimilar to wild-type R1 and D3 ESCs with a correlation coefficient (R2) of 0.92 (Figure 3A). Both OSK and OSKT iPS cells bore closer resemblance to ESCs but were indistinguishable from each other; R2 = 0.94. Hence, global expression profiling was not sufficiently sensitive in detecting differences between these clones. Closer examination of specific gene level alterations, however, revealed key differences (Figure 3B; Figure S11). We compared the pluripotency-associated gene levels among OSK, OSKT iPS and ES cells. The majority of these genes such as Sall4, Tcf3, Sox2, Zfx, Lin28, Utf1 and Zic3 were non-distinguishing between OSKT and OSK cells as their levels were similar. Surprisingly, a small subset of distinguishing features appeared to define OSKT from OSK cells. Oct4, Nanog, Gdf3, Dppa4 and Tbx3 levels in OSKT cells were equivalent to ESCs, but significantly reduced in OSK cells. This suggests that exogenous Tbx3 may be crucial for assisting in re-establishing proper levels of certain ESC factors critical for the induction of pluripotency that cannot be completely achieved with OSK alone.

Bottom Line: Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency.Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1.This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Developmental Biology, Genome Institute of Singapore, 138672, Singapore.

ABSTRACT
Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

Show MeSH
Related in: MedlinePlus