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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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Global gene expression profiling reveals Tbx3 aids cell fusion-mediated reprogramming. (A) Modified ESCs with Nanog over-expression (OE) or Tcf3 RNAi were fused with MEFs to generate tetraploid ESC/MEF hybrids resistant to neomycin and puromycin. (B) Nanog OE, Tbx3 OE and Tcf3 RNAi enhanced cell fusion-mediated reprogramming of MEFs. Representative examples illustrate the emergence of ESC/MEF hybrid colonies. Control ESC fusion with MEFs resulted in an average of one per experiment whereas Tcf3 RNAi, Nanog OE or Tbx3 OE ESCs produced numerous hybrid clones. (C) Nanog OE ESCs were efficient in reprogramming MEFs, generating 13 colonies, followed by Tcf3 RNAi (10) and Tbx3 OE (4.5). The numbers represent the average of four independent fusion experiments. * denotes significantly different from vector, + denotes significantly different from control shRNA; error bars represent s.e.m. (D) The heat-map shows all genes which were increased in treated ESCs compared to controls. Tbx3 was among the most highly up-regulated genes in Nanog OE and Tcf3 RNAi ESCs. The left-most column in red indicates direct gene targets of Nanog or Tcf3 based on the ChIP-PET21 and ChIP-chip13 databases respectively. (E) RNAi knockdown of Tbx3 in ESCs led to a loss of self-renewal and induced differentiation. Scale bar = 100 μm. (F) Enrichment of Tcf3 and Nanog occupancy on the Tbx3 gene, as measured by ChIP-qPCR.
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Figure 1: Global gene expression profiling reveals Tbx3 aids cell fusion-mediated reprogramming. (A) Modified ESCs with Nanog over-expression (OE) or Tcf3 RNAi were fused with MEFs to generate tetraploid ESC/MEF hybrids resistant to neomycin and puromycin. (B) Nanog OE, Tbx3 OE and Tcf3 RNAi enhanced cell fusion-mediated reprogramming of MEFs. Representative examples illustrate the emergence of ESC/MEF hybrid colonies. Control ESC fusion with MEFs resulted in an average of one per experiment whereas Tcf3 RNAi, Nanog OE or Tbx3 OE ESCs produced numerous hybrid clones. (C) Nanog OE ESCs were efficient in reprogramming MEFs, generating 13 colonies, followed by Tcf3 RNAi (10) and Tbx3 OE (4.5). The numbers represent the average of four independent fusion experiments. * denotes significantly different from vector, + denotes significantly different from control shRNA; error bars represent s.e.m. (D) The heat-map shows all genes which were increased in treated ESCs compared to controls. Tbx3 was among the most highly up-regulated genes in Nanog OE and Tcf3 RNAi ESCs. The left-most column in red indicates direct gene targets of Nanog or Tcf3 based on the ChIP-PET21 and ChIP-chip13 databases respectively. (E) RNAi knockdown of Tbx3 in ESCs led to a loss of self-renewal and induced differentiation. Scale bar = 100 μm. (F) Enrichment of Tcf3 and Nanog occupancy on the Tbx3 gene, as measured by ChIP-qPCR.

Mentions: We speculated that iPS cell-reprogramming factors may share common characteristics with pluripotency-associated genes whose perturbed levels in ESCs confer resistance to differentiation. Previous studies have shown that mouse ESCs over-expressing Nanog are resistant to differentiation11, express higher levels of pluripotency-associated genes, and are more effective at reprogramming somatic cells through cell fusion12. Another transcription factor Tcf3 when depleted in ESCs limits their differentiation ability, and upregulates the expression of pluripotency markers that includes Oct4, Sox2, Nanog and Sall413. As both Nanog and Tcf3 regulate each other, and are core features of the ESC transcriptional network, we speculated that similar to Nanog over-expressing ESCs, the loss of Tcf3 may enhance fusion-mediated reprogramming of somatic hybrid cells. To test this, we used polyethyleneglycol (PEG) to generate duo drug-resistant fusion hybrids between Nanog over-expressing (OE) or Tcf3 RNAi ESCs that were neomycin-resistant (NeoR) and primary MEFs that were puromycin-resistant (PuroR) (Figure 1A). Consistent with previous observations, Nanog OE ESCs showed enhanced reprogramming efficiency (Figure 1B & C). Using Tcf3-deficient ESC lines, a significant increase in the number of hybrid clones was also observed (Figure 1B & C). Karyotype analysis confirmed that these were tetraploid (Figure S1). The hybrids possess properties similar to the parental modified ESC lines, including their response to the lack of leukemia inhibitory factor (LIF) and epigenetic reprogramming of the Nanog promoter (Figure S2 & S3). We eliminated the possibility that improvements in reprogramming frequency was attributed to increased cell fusion events12 (Figure S4).


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)

Global gene expression profiling reveals Tbx3 aids cell fusion-mediated reprogramming. (A) Modified ESCs with Nanog over-expression (OE) or Tcf3 RNAi were fused with MEFs to generate tetraploid ESC/MEF hybrids resistant to neomycin and puromycin. (B) Nanog OE, Tbx3 OE and Tcf3 RNAi enhanced cell fusion-mediated reprogramming of MEFs. Representative examples illustrate the emergence of ESC/MEF hybrid colonies. Control ESC fusion with MEFs resulted in an average of one per experiment whereas Tcf3 RNAi, Nanog OE or Tbx3 OE ESCs produced numerous hybrid clones. (C) Nanog OE ESCs were efficient in reprogramming MEFs, generating 13 colonies, followed by Tcf3 RNAi (10) and Tbx3 OE (4.5). The numbers represent the average of four independent fusion experiments. * denotes significantly different from vector, + denotes significantly different from control shRNA; error bars represent s.e.m. (D) The heat-map shows all genes which were increased in treated ESCs compared to controls. Tbx3 was among the most highly up-regulated genes in Nanog OE and Tcf3 RNAi ESCs. The left-most column in red indicates direct gene targets of Nanog or Tcf3 based on the ChIP-PET21 and ChIP-chip13 databases respectively. (E) RNAi knockdown of Tbx3 in ESCs led to a loss of self-renewal and induced differentiation. Scale bar = 100 μm. (F) Enrichment of Tcf3 and Nanog occupancy on the Tbx3 gene, as measured by ChIP-qPCR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2901797&req=5

Figure 1: Global gene expression profiling reveals Tbx3 aids cell fusion-mediated reprogramming. (A) Modified ESCs with Nanog over-expression (OE) or Tcf3 RNAi were fused with MEFs to generate tetraploid ESC/MEF hybrids resistant to neomycin and puromycin. (B) Nanog OE, Tbx3 OE and Tcf3 RNAi enhanced cell fusion-mediated reprogramming of MEFs. Representative examples illustrate the emergence of ESC/MEF hybrid colonies. Control ESC fusion with MEFs resulted in an average of one per experiment whereas Tcf3 RNAi, Nanog OE or Tbx3 OE ESCs produced numerous hybrid clones. (C) Nanog OE ESCs were efficient in reprogramming MEFs, generating 13 colonies, followed by Tcf3 RNAi (10) and Tbx3 OE (4.5). The numbers represent the average of four independent fusion experiments. * denotes significantly different from vector, + denotes significantly different from control shRNA; error bars represent s.e.m. (D) The heat-map shows all genes which were increased in treated ESCs compared to controls. Tbx3 was among the most highly up-regulated genes in Nanog OE and Tcf3 RNAi ESCs. The left-most column in red indicates direct gene targets of Nanog or Tcf3 based on the ChIP-PET21 and ChIP-chip13 databases respectively. (E) RNAi knockdown of Tbx3 in ESCs led to a loss of self-renewal and induced differentiation. Scale bar = 100 μm. (F) Enrichment of Tcf3 and Nanog occupancy on the Tbx3 gene, as measured by ChIP-qPCR.
Mentions: We speculated that iPS cell-reprogramming factors may share common characteristics with pluripotency-associated genes whose perturbed levels in ESCs confer resistance to differentiation. Previous studies have shown that mouse ESCs over-expressing Nanog are resistant to differentiation11, express higher levels of pluripotency-associated genes, and are more effective at reprogramming somatic cells through cell fusion12. Another transcription factor Tcf3 when depleted in ESCs limits their differentiation ability, and upregulates the expression of pluripotency markers that includes Oct4, Sox2, Nanog and Sall413. As both Nanog and Tcf3 regulate each other, and are core features of the ESC transcriptional network, we speculated that similar to Nanog over-expressing ESCs, the loss of Tcf3 may enhance fusion-mediated reprogramming of somatic hybrid cells. To test this, we used polyethyleneglycol (PEG) to generate duo drug-resistant fusion hybrids between Nanog over-expressing (OE) or Tcf3 RNAi ESCs that were neomycin-resistant (NeoR) and primary MEFs that were puromycin-resistant (PuroR) (Figure 1A). Consistent with previous observations, Nanog OE ESCs showed enhanced reprogramming efficiency (Figure 1B & C). Using Tcf3-deficient ESC lines, a significant increase in the number of hybrid clones was also observed (Figure 1B & C). Karyotype analysis confirmed that these were tetraploid (Figure S1). The hybrids possess properties similar to the parental modified ESC lines, including their response to the lack of leukemia inhibitory factor (LIF) and epigenetic reprogramming of the Nanog promoter (Figure S2 & S3). We eliminated the possibility that improvements in reprogramming frequency was attributed to increased cell fusion events12 (Figure S4).

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