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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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OSKT iPS cells show enhanced germ-line contribution and transmission. The iPS clone ID and the types of analyses that were performed is summarized in Table S7. (A) Representative photos showing the quantitative contribution and spatial distribution of GFP+ cells generated with different reprogramming factor combinations in the gonads of chimeric embryos. (B) Tabulation comparing the contribution of GFP+ iPS cell-derived germ cells to the gonads of chimeric fetuses and their spatial distribution with different combinations of factors. OSKT iPS cells were most effective at colonizing the gonads, compared to OSK and OSE iPS cells. * denotes significantly different from OSK, p<0.01. At least eleven independent sets of microinjections were performed for OSK versus OSKT comparison. (C) Representative photos showing the contribution of iPS cells to chimeric coat and the production of F2 offspring after crossing with albino mice. Black offspring indicates germ-line transmission of iPS cells. (D) Table summarizing the germ-line transmission frequency for iPS cells generated with the different combination of factors. For each combination, between two to six iPS cell lines were used to obtain live chimeras, of which, at least one male from each combination were crossed with an albino female to determine the frequency of black F2. * denotes significantly different from OSK and OSE, p<0.005 and p<0.001 respectively. (E) Live birth animals obtained from two OSKT iPS cell lines tested by tetraploid complementation.
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Figure 4: OSKT iPS cells show enhanced germ-line contribution and transmission. The iPS clone ID and the types of analyses that were performed is summarized in Table S7. (A) Representative photos showing the quantitative contribution and spatial distribution of GFP+ cells generated with different reprogramming factor combinations in the gonads of chimeric embryos. (B) Tabulation comparing the contribution of GFP+ iPS cell-derived germ cells to the gonads of chimeric fetuses and their spatial distribution with different combinations of factors. OSKT iPS cells were most effective at colonizing the gonads, compared to OSK and OSE iPS cells. * denotes significantly different from OSK, p<0.01. At least eleven independent sets of microinjections were performed for OSK versus OSKT comparison. (C) Representative photos showing the contribution of iPS cells to chimeric coat and the production of F2 offspring after crossing with albino mice. Black offspring indicates germ-line transmission of iPS cells. (D) Table summarizing the germ-line transmission frequency for iPS cells generated with the different combination of factors. For each combination, between two to six iPS cell lines were used to obtain live chimeras, of which, at least one male from each combination were crossed with an albino female to determine the frequency of black F2. * denotes significantly different from OSK and OSE, p<0.005 and p<0.001 respectively. (E) Live birth animals obtained from two OSKT iPS cell lines tested by tetraploid complementation.

Mentions: We then investigated whether OSKT cells were of higher quality than OSK and OSE cells (Figure S12; summarized in Table S7). We selected numerous iPS cell lines generated from each combination which showed homogeneous activation of Oct4-GFP within each colony: OSKT #1,2,4,6,11-22; OSK #1-3,12-14,16-22; OSE #1,2,9. These lines were derived from four independent transduction experiments performed at different times to eliminate any biasness in clonal selection that may arise from the stochastic behavior of individual clones. Induced-PS cells were then injected into 4-8 cell embryos and cultured in vitro to blastocysts (Figure S13). There was no difference in the maturation efficiency (~95%) between the three iPS cell types (data not shown). All blastocysts transplanted into the surrogate female mice initially had shown robust contribution of GFP+ cells to the inner cell mass (ICM) (Figure S13) and live chimeras were obtained. As evaluated by coat color, all OSKT and OSK chimeras showed coats with varying density of black fur, signifying iPS cell contribution; whereas OSE chimeras clearly had lesser black coat (Figure 4C, top panels), indicating OSE cells tend to contribute poorly to chimerism.


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)

OSKT iPS cells show enhanced germ-line contribution and transmission. The iPS clone ID and the types of analyses that were performed is summarized in Table S7. (A) Representative photos showing the quantitative contribution and spatial distribution of GFP+ cells generated with different reprogramming factor combinations in the gonads of chimeric embryos. (B) Tabulation comparing the contribution of GFP+ iPS cell-derived germ cells to the gonads of chimeric fetuses and their spatial distribution with different combinations of factors. OSKT iPS cells were most effective at colonizing the gonads, compared to OSK and OSE iPS cells. * denotes significantly different from OSK, p<0.01. At least eleven independent sets of microinjections were performed for OSK versus OSKT comparison. (C) Representative photos showing the contribution of iPS cells to chimeric coat and the production of F2 offspring after crossing with albino mice. Black offspring indicates germ-line transmission of iPS cells. (D) Table summarizing the germ-line transmission frequency for iPS cells generated with the different combination of factors. For each combination, between two to six iPS cell lines were used to obtain live chimeras, of which, at least one male from each combination were crossed with an albino female to determine the frequency of black F2. * denotes significantly different from OSK and OSE, p<0.005 and p<0.001 respectively. (E) Live birth animals obtained from two OSKT iPS cell lines tested by tetraploid complementation.
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Related In: Results  -  Collection

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Figure 4: OSKT iPS cells show enhanced germ-line contribution and transmission. The iPS clone ID and the types of analyses that were performed is summarized in Table S7. (A) Representative photos showing the quantitative contribution and spatial distribution of GFP+ cells generated with different reprogramming factor combinations in the gonads of chimeric embryos. (B) Tabulation comparing the contribution of GFP+ iPS cell-derived germ cells to the gonads of chimeric fetuses and their spatial distribution with different combinations of factors. OSKT iPS cells were most effective at colonizing the gonads, compared to OSK and OSE iPS cells. * denotes significantly different from OSK, p<0.01. At least eleven independent sets of microinjections were performed for OSK versus OSKT comparison. (C) Representative photos showing the contribution of iPS cells to chimeric coat and the production of F2 offspring after crossing with albino mice. Black offspring indicates germ-line transmission of iPS cells. (D) Table summarizing the germ-line transmission frequency for iPS cells generated with the different combination of factors. For each combination, between two to six iPS cell lines were used to obtain live chimeras, of which, at least one male from each combination were crossed with an albino female to determine the frequency of black F2. * denotes significantly different from OSK and OSE, p<0.005 and p<0.001 respectively. (E) Live birth animals obtained from two OSKT iPS cell lines tested by tetraploid complementation.
Mentions: We then investigated whether OSKT cells were of higher quality than OSK and OSE cells (Figure S12; summarized in Table S7). We selected numerous iPS cell lines generated from each combination which showed homogeneous activation of Oct4-GFP within each colony: OSKT #1,2,4,6,11-22; OSK #1-3,12-14,16-22; OSE #1,2,9. These lines were derived from four independent transduction experiments performed at different times to eliminate any biasness in clonal selection that may arise from the stochastic behavior of individual clones. Induced-PS cells were then injected into 4-8 cell embryos and cultured in vitro to blastocysts (Figure S13). There was no difference in the maturation efficiency (~95%) between the three iPS cell types (data not shown). All blastocysts transplanted into the surrogate female mice initially had shown robust contribution of GFP+ cells to the inner cell mass (ICM) (Figure S13) and live chimeras were obtained. As evaluated by coat color, all OSKT and OSK chimeras showed coats with varying density of black fur, signifying iPS cell contribution; whereas OSE chimeras clearly had lesser black coat (Figure 4C, top panels), indicating OSE cells tend to contribute poorly to chimerism.

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