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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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Generation of iPS cells with Oct4, Sox2, Klf4 and Tbx3 retroviral transduction. (A) OSKC and OSKCT induced ~300 ESC-like colonies with ~10% expressing GFP, whereas OSKT induced an average of 38 compared to 26 ESC-like colonies from OSK, with 89% and 74% GFP activation respectively. Colonies were assessed and counted on day 16. Data represents the average of three independent transduction experiments. * denotes significantly different from OSK; p<0.05; error bars represent s.e.m. (B) OSK and OSKT iPS cell clones showed tight, domed-shaped ESC-like morphology and uniform GFP expression throughout the colony, whereas OSKC clones appeared as flattened, transformed cells with sparse GFP expression. Scale bar = 100 μm. (C) OSKT induced the activation of Oct4-GFP transgene in iPS derived from primary MEFs at 9-10 days post-infection while OSK and OSKC combinations required 14 days observed in four independent transduction experiment.
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Figure 2: Generation of iPS cells with Oct4, Sox2, Klf4 and Tbx3 retroviral transduction. (A) OSKC and OSKCT induced ~300 ESC-like colonies with ~10% expressing GFP, whereas OSKT induced an average of 38 compared to 26 ESC-like colonies from OSK, with 89% and 74% GFP activation respectively. Colonies were assessed and counted on day 16. Data represents the average of three independent transduction experiments. * denotes significantly different from OSK; p<0.05; error bars represent s.e.m. (B) OSK and OSKT iPS cell clones showed tight, domed-shaped ESC-like morphology and uniform GFP expression throughout the colony, whereas OSKC clones appeared as flattened, transformed cells with sparse GFP expression. Scale bar = 100 μm. (C) OSKT induced the activation of Oct4-GFP transgene in iPS derived from primary MEFs at 9-10 days post-infection while OSK and OSKC combinations required 14 days observed in four independent transduction experiment.

Mentions: We postulated that Tbx3 may also improve the reprogramming efficiency or the quality of iPS cells. Retroviral infection of MEFs bearing the Oct4-GFP transgene with OSKC induced ~300 ESC-like colonies per 5 × 104 starting cells (Figure 2A). However, only ~10% of these showed activation of the transgene. The addition of Tbx3 (OSKCT) did not increase the frequency of GFP+ colony numbers (Figure 2A). With three factors (OSK), the total number of ESC-like colonies was dramatically reduced but 74% of OSK colonies expressed GFP (Figure 2A). The addition of Tbx3 (OSKT) improved the colony count (~38 on average) when compared with OSK (~26), and the percentage of GFP+ colonies also significantly increased to 89% (Figure 2A). Other qualitative differences between iPS cells obtained with different factor combinations were observed. OSKC iPS cell colonies were difficult to distinguish amongst transformed and partially reprogrammed cells that did not show GFP expression. OSK and OSKT iPS cell colonies were morphologically similar, with uniform GFP expression within individual colonies (Figure 2B). However, while the activation of Oct4 typically required 14 days post-infection with OSK and OSKC, the use of OSKT took 9-10 days, suggesting that Tbx3 accelerated the reprogramming process (Figure 2C). The efficiency of isolating stable iPS cell lines from GFP+ colonies was similar between OSK and OSKT transduction, but almost two-fold higher than OSKC and OSKCT (Figure S7). For all the iPS cell lines obtained, we performed PCR analyses and confirmed genomic integration of the respective transduced genes (Figure S8). Molecular characterization of iPS cells from OSKT confirmed these were alkaline phosphatase-positive, expressed Nanog, Sox2 and SSEA1 (Figure S9). They also form teratomas comprised of multiple differentiated cell types when xenografted into SCID mice (Figure S10).


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)

Generation of iPS cells with Oct4, Sox2, Klf4 and Tbx3 retroviral transduction. (A) OSKC and OSKCT induced ~300 ESC-like colonies with ~10% expressing GFP, whereas OSKT induced an average of 38 compared to 26 ESC-like colonies from OSK, with 89% and 74% GFP activation respectively. Colonies were assessed and counted on day 16. Data represents the average of three independent transduction experiments. * denotes significantly different from OSK; p<0.05; error bars represent s.e.m. (B) OSK and OSKT iPS cell clones showed tight, domed-shaped ESC-like morphology and uniform GFP expression throughout the colony, whereas OSKC clones appeared as flattened, transformed cells with sparse GFP expression. Scale bar = 100 μm. (C) OSKT induced the activation of Oct4-GFP transgene in iPS derived from primary MEFs at 9-10 days post-infection while OSK and OSKC combinations required 14 days observed in four independent transduction experiment.
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Related In: Results  -  Collection

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Figure 2: Generation of iPS cells with Oct4, Sox2, Klf4 and Tbx3 retroviral transduction. (A) OSKC and OSKCT induced ~300 ESC-like colonies with ~10% expressing GFP, whereas OSKT induced an average of 38 compared to 26 ESC-like colonies from OSK, with 89% and 74% GFP activation respectively. Colonies were assessed and counted on day 16. Data represents the average of three independent transduction experiments. * denotes significantly different from OSK; p<0.05; error bars represent s.e.m. (B) OSK and OSKT iPS cell clones showed tight, domed-shaped ESC-like morphology and uniform GFP expression throughout the colony, whereas OSKC clones appeared as flattened, transformed cells with sparse GFP expression. Scale bar = 100 μm. (C) OSKT induced the activation of Oct4-GFP transgene in iPS derived from primary MEFs at 9-10 days post-infection while OSK and OSKC combinations required 14 days observed in four independent transduction experiment.
Mentions: We postulated that Tbx3 may also improve the reprogramming efficiency or the quality of iPS cells. Retroviral infection of MEFs bearing the Oct4-GFP transgene with OSKC induced ~300 ESC-like colonies per 5 × 104 starting cells (Figure 2A). However, only ~10% of these showed activation of the transgene. The addition of Tbx3 (OSKCT) did not increase the frequency of GFP+ colony numbers (Figure 2A). With three factors (OSK), the total number of ESC-like colonies was dramatically reduced but 74% of OSK colonies expressed GFP (Figure 2A). The addition of Tbx3 (OSKT) improved the colony count (~38 on average) when compared with OSK (~26), and the percentage of GFP+ colonies also significantly increased to 89% (Figure 2A). Other qualitative differences between iPS cells obtained with different factor combinations were observed. OSKC iPS cell colonies were difficult to distinguish amongst transformed and partially reprogrammed cells that did not show GFP expression. OSK and OSKT iPS cell colonies were morphologically similar, with uniform GFP expression within individual colonies (Figure 2B). However, while the activation of Oct4 typically required 14 days post-infection with OSK and OSKC, the use of OSKT took 9-10 days, suggesting that Tbx3 accelerated the reprogramming process (Figure 2C). The efficiency of isolating stable iPS cell lines from GFP+ colonies was similar between OSK and OSKT transduction, but almost two-fold higher than OSKC and OSKCT (Figure S7). For all the iPS cell lines obtained, we performed PCR analyses and confirmed genomic integration of the respective transduced genes (Figure S8). Molecular characterization of iPS cells from OSKT confirmed these were alkaline phosphatase-positive, expressed Nanog, Sox2 and SSEA1 (Figure S9). They also form teratomas comprised of multiple differentiated cell types when xenografted into SCID mice (Figure S10).

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