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Keywords:

  • TBX3;
  • Human embryonic stem cells;
  • Self-renewal;
  • Differentiation;
  • Neuroepithelial

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self-renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both NFκBIB and p14ARF, known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, and RAX). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both hESCs and mouse ESCs. STEM Cells2012;30:2152–2163


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

T-box 3 (Tbx3) is a member of the T-box gene family. The T-box family of transcription factors plays an important role in regulating early developmental processes and in regulating gene expression networks involved in specifying cell lineage [1–3]. Homozygous mutations of Tbx3 are embryonic lethal in mice suggesting that Tbx3 is important during the earliest phases of embryo development [4]. In humans, heterozygous mutations of TBX3 are sufficient to cause ulnar mammary syndrome characterized by defects within the limbs, apocrine, and mammary gland, similar to those in mouse models [4–11].

In addition to its role in regulating early embryo and mammary gland development, Tbx3 has been identified as a key regulator of mouse embryonic stem cell (ESC) pluripotency and differentiation. In mouse ESCs undergoing retinoic acid (RA)-induced differentiation, Tbx3 was significantly downregulated along with known pluripotency regulators Oct4 and Nanog [12]. Knockdown of Tbx3 resulted in the loss of pluripotency and differentiation of mouse ESCs, suggesting that Tbx3 is necessary to maintain self-renewal [12]. While overexpression of Tbx3 was found to be sufficient to maintain mouse ESCs in their undifferentiated state in the absence of leukemia inhibitor factor (LIF) [13], Tbx3 overexpression also induced differentiation into ExEn through direct regulation of Gata6 [14]. A more recent study has shown that Tbx3 not only maintains mouse ESC self-renewal but also plays an essential role in their differentiation [14]. Knockdown of Tbx3, during mouse embryoid body (EB) formation, prevented ExEn differentiation, while enhancing ectoderm and trophectoderm differentiation [14]. In addition, expression of Tbx3 during somatic cell reprogramming has been shown to improve the overall quality of induced pluripotent stem (iPS) cells [15]. These studies confirm that Tbx3 is not only necessary to maintain self-renewal but also plays a role in regulating differentiation of mouse ESCs into ExEn.

Although accumulating evidence suggests Tbx3 plays roles in regulating the self-renewal and differentiation of mouse ESCs, the function of TBX3 in regulating human ESC (hESC) self-renewal and differentiation remains largely unexplored. Molecular analyses suggest that hESCs and mouse ESCs represent distinct pluripotent states, in which mouse ESCs are considered to be at a more naïve pluripotent state and as a result have very different biological properties [16, 17]. This is reflected by the fact that the same transcriptional and signaling pathways regulate mouse and hESC pluripotency differently. Addition of LIF or bone morphogenetic proteins (BMPs) to mouse ESCs is necessary to maintain pluripotency while these same factors cause differentiation in hESCs [18–22]. Due to the differences between their pluripotent states, TBX3 may function very differently in hESCs than in mouse ESCs. Unraveling hESC transcriptional circuitry is fundamental for understanding ESC differentiation and specification in early human development. It is also a key step for the development of improved methods to derive, culture, and differentiate hESCs into cells of a specific developmental pathway for potential therapeutic use.

In this study, we generated TBX3 overexpressing and TBX3 knockdown hESC lines to investigate the role of TBX3 in regulating hESC self-renewal and differentiation. In contrast to mouse ESC studies, we found that neither overexpression nor knockdown of TBX3 was able to attenuate self-renewal ability to induce differentiation in undifferentiated hESCs. Instead, overexpression of TBX3 promoted stem cell proliferation possibly by repressing the expression of cell cycle regulators, NFκBIB and p14-alternate reading frame (p14ARF). Also, knockdown of TBX3 during differentiation reduced neural rosette formation as well as the expression of neuroepithelial and neuroectoderm markers (FOXG1, PAX6, LHX2, and RAX). Our study suggests TBX3 stimulates hESC proliferation and promotes neuroepithelial differentiation. Our data demonstrate that TBX3 plays distinct roles in hESCs and mouse ESCs in regulating self-renewal and differentiation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Maintenance of hESCs

hESCs (H9, H1) were obtained from WiCell (Madison, WI, http://www.wicell.org/). Cells were maintained on mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer and fed daily with hESC medium (knockout-Dulbecco's modified Eagle's medium [DMEM] supplemented with 20% knockout serum replacement, 1% Glutamax, 1% nonessential amino acids, 6 ng/ml basic fibroblast growth factor [bFGF] [all from Life Technologies, Grand Island, NY, http://www.invitrogen.com], and 0.1 mM β-mercaptoethanol [Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com]). Cells were passaged enzymatically every 4–6 days with 1 mg/ml collagenase type IV (Life Technologies, Grand Island, NY, http://www.invitrogen.com) and seeded onto MEF feeder layers. When appropriate, hESCs were also cultured on matrigel (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) and fed daily with conditioned medium (CM) supplemented with 6 ng/ml bFGF.

hESC Differentiation

To induce differentiation of hESCs as EBs, colonies of undifferentiated hESCs were treated with 1 mg/ml collagenase type IV for 10 minutes and dissociated cells into clumps. Clumps of undifferentiated hESCs were cultured in suspension and fed ESC medium without bFGF (differentiation medium) every other day for 5 or 7 days. On the fifth or seventh day, EBs were collected and plated onto 0.2% gelatin-coated plates. Plated EBs were fed every other day with differentiation medium or differentiation medium supplemented with 10% fetal bovine serum (FBS), 100 ng/ml BMP4, or 1 μM RA for an additional 11 or 14 days.

Directed Neuroepithelial Differentiation

hESCs were induced to differentiate toward neuroepithelia as previously described by Shi et al. [23]. Briefly, hESCs (H9) were passaged using 1 mg/ml collagenase and plated on Matrigel-coated 12-well plates in MEF CM supplemented with 10 ng/ml bFGF2. Once the cells reached 90% confluence, neural induction was initiated by changing the culture medium to one that supports neural induction; a 1:1 mixture of N2- and B27-containing media referred to as 3N medium. N2 medium consisted of DMEM/F12, N2 (Life Technologies, Grand Island, NY, http://www.invitrogen.com), 5 μg/ml insulin, 1 mM L-glutamine, 100 μM nonessential amino acids, 100 μM 2-mercaptoethanol, 50 U/ml penicillin, and 50 mg/ml streptomycin. B27 medium consisted of Neurobasal (Life Technologies, Grand Island, NY, http://www.invitrogen.com), B27 (Life Technologies, Grand Island, NY, http://www.invitrogen.com), 200 mM glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin. 3N medium was supplemented with 1 μM RA, 1 μM Dorsomorphin (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), and 10 μM SB431542 (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) [23]. Cells were fed every other day for 14 days. The efficiency of neural induction was monitored by the appearance of cells with characteristic neuroepithelial cell morphology (neural rosette formation) and expression of established neuroepithelial markers, PAX6, NESTIN, and the apical redistribution of ZO-1.

Plasmids

For overexpression constructs, we digested the FUGW-PGK-eGFP-Puromycin plasmid with restriction enzymes, BamHI and BsrGI, to excise enhanced green fluorescence protein (eGFP). Digested plasmid was run on a 1% agarose gel and purified using Qiagen Gel Extraction Kit (Qiagen, Valencia, CA, http://www.qiagen.com) according to manufacturer's protocol. Next, the full length cDNA of TBX3, along with a myc tag, was PCR amplified out of pcDNA-myc-TBX3, digested with restriction enzymes (BamHI and BsrGI), and ligated into the FUGW-PGK-Puromycin plasmid. Positive clones were verified by restriction enzyme digestion and sequencing. The control plasmid was constructed in a similar fashion except only the myc tag was ligated into the FUGW-PGK-Puromycin plasmid. For TBX3 knockdown constructs, the plko.1-control and various plko.1-TBX3shRNA plasmids were a generous gift from Dr. Anand Ganesan.

Verification of Plko.1-TBX3 shRNA Lentiviral Vectors

Nearly confluent MCF7 cells, overexpressing TBX3-eGFP fusion protein, were transfected with 6 μg of control or TBX3 shRNA lentiviral vectors using Lipofectamine 2000 (Life Technologies, Grand Island, NY, http://www.invitrogen.com) transfection reagent, according to manufacturer's protocol. Seventy-two hours after transfection, cell lysates were harvested and used to perform Western blot to verify knockdown.

Western Blotting

Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a NuPAGE 4%–12% gradient Bis-Tris gel (Life Technologies, Grand Island, NY, http://www.invitrogen.com) and electroblotted onto a polyvinylidene difluoride membrane (Life Technologies, Grand Island, NY, http://www.invitrogen.com). Membranes were incubated in 5% blocking buffer (non-fat dry milk in phosphate buffered saline (PBS) containing 0.1% Tween 20) for 3 hours at room temperature. Membranes were probed with mouse anti-myc (1:2,000; Clontech Laboratories, Mountain View, CA, http://www.clontech.com) overnight at 4°C. Membranes were then washed three times with PBS-T and incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; Thermo Scientific, Rockford, IL, http://www.pierce-antibodies.com) for 1.5 hours at 4°C. HRP was detected using ECL Western Blotting Detection Reagents (GE Healthcare Biosciences, Pittsburgh, PA, http://www.gelifesciences.com).

Lentivirus Production

Lentivirus was produced by transient cotransfection of three plasmids into 293T cells. Briefly, 293T cells were plated onto poly(D-lysine)-coated plates at approximately 80%–90% confluence. 293T cells were transfected using Lipofectamine 2000 reagent (Life Technologies, Grand Island, NY, http://www.invitrogen.com), according to the manufacturer's protocol. A total of 16 μg of plasmid DNA per 100 mm dish was used: 2 μg of the VSV-G envelope plasmid, pMD.G, 8 μg of the packaging plasmid, pCMVR8.91, and 6 μg of the Lentiviral transducing vector. The medium was replaced 24 hours after transfection with virus-collecting medium (DMEM supplemented with 1% FBS). The viral supernatant was collected at 48 and 72 hours after transfection and filtered through 0.45 μm filters. Virus was concentrated using the Amicon Ultra-15 Centrifugal filter device (Millipore, Billerica, MA, http://www.millipore.com) as recommended by the manufacturer and immediately added to cells.

Transduction of hESCs to Create TBX3 Overexpressing and Knockdown Cell Lines

Twenty-four hours prior to transduction, hESCs were mechanically passaged from MEFs onto matrigel to eliminate MEFs during transduction. The following day, concentrated lentivirus was added to fresh CM supplemented with 6 μg/ml polybrene and added to the cells. hESCs were transduced twice, once at the 48 hour viral harvest and once at the 72 hour viral harvest. Forty-eight hours after the second transduction, puromycin (1 μg/ml) was used to select for transduced ESCs. Single clones were picked, expanded, and verified for either TBX3 overexpression or knockdown.

Generation of H9-hOCT4-eGFP Cell Line

The hOCT4-eGFP plasmid was a generous gift from Dr. Wei Cui laboratory. hESCs were transfected as previously described [24]. Briefly, 24 hours prior to transfection, hESCs were mechanically passaged onto matrigel. The following day, 10 μg of ApaLI linearized hOCT4-eGFP plasmid was transfected into hESCs using Fugene 6 (Roche, Indianapolis, IN, http://www.roche.com/), following manufacturer's instructions. The DNA/Fugene ratio of 1:1.5 was used. Forty-eight hours later, transfected cells were selected for using G418 at 200 μg/ml. Single clones were picked and expanded.

Transient TBX3 Overexpression Experiments

Prior to transduction, OCT4-eGFP hESCs were mechanically passaged onto matrigel and then transduced at 24 and 48 hours after plating, with myc or myc-TBX3 lentivirus. Six days after initial transduction, cells were fixed with 4% paraformaldehyde.

Quantitative Real-Time Polymerase Chain Reaction Analysis

RNA was isolated and purified using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, http://www.qiagen.com), according to the manufacturer's recommendations. SYBR Green quantitative real-time polymerase chain reactions (qRT-PCRs) were performed in triplicate for each primer set using the ABI 7900HT Sequence Detection System (Applied Biosystems, Carlsbad, CA, https://products.appliedbiosystems.com). Primers were designed to span introns or at exon junctions. To ensure specificity of PCR, melt curve analyses were performed at the end of all PCRs. Gene expression levels were normalized to GAPDH and then analyzed using the 2inline image method.

Immunocytochemistry

For immunocytochemistry analyses, cells were fixed with 4% paraformaldehyde in PBS for 20 minutes. Fixed cells were permeabilized with 0.1% Triton X-100 and 10% goat serum in PBS (10% blocking buffer) for 1 hour at room temperature. The cells were incubated with primary antibodies in 10% blocking buffer overnight at 4°C, followed by incubation with appropriate Alexa Fluor 488 or 594-conjugated secondary antibodies (1:400; Life Technologies, Grand Island, NY, http://www.invitrogen.com) in 10% blocking buffer for 1.5 hours at 4°C. Cell nuclei were visualized by 4′-6-diamidino-2-phenylindole (DAPI, 1:400) staining. Immunofluorescence images were captured on a Nikon Eclipse Ti Inverted Microscope. Antibodies against the following proteins were used: myc (1:200; Clontech Laboratories, Mountain View, CA, http://www.clontech.com), TBX3 (1:50; Life Technologies, Grand Island, NY, http://www.invitrogen.com), OCT4 (1:200; Abcam, Cambridge, MA, http://www.abcam.com), NANOG (1:100; Cell Signaling, Danvers, MA http://www.cellsignal.com/), PAX6 (1:300; Covance, Princeton, NJ http://www.covance.com), NESTIN (1:200; Life Technologies, Grand Island, NY, http://www.invitrogen.com); SOX1 (1:300; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), and ZO-1 (1:300; Life Technologies, Grand Island, NY, http://www.invitrogen.com).

Mitotic Index

hESCs were fixed with 4% paraformaldehyde in PBS for 20 minutes. Fixed cells were permeabilized with 0.1% Triton X-100 and incubated with DAPI (1:400) for 1 hour at room temperature. DAPI images were captured on a Nikon Eclipse Ti Inverted Microscope. The Image J software was used to count mitotic nuclei and total number of nuclei present. To calculate the mitotic index, the number of mitotic nuclei was divided by the total number of nuclei. Approximately 2,000–5,000 cells were scored.

5-Bromo-2-deoxyuridine Incorporation Assay

Control and TBX3 overexpressing hESC proliferation was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation. Cells were seeded onto 12-well matrigel-coated plates in MEF-CM supplemented with 10 ng/ml bFGF and allowed to grow for 4 days. On the fourth day, BrdU was diluted into fresh MEF-CM to a final concentration of 10 μM and incubated at 37°C for 4 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. Fixed cells were washed, treated with 1.5 M HCl for 30 minutes at room temperature, and washed once more. BrdU that had been incorporated into newly synthesized DNA was detected by immunocytochemistry as described above (as described in Immunocytochemistry). Mouse anti-BrdU (1:1,000; Millipore, Billerica, MA, http://www.millipore.com) and goat anti-mouse Alexa Fluor 594-conjugated secondary antibodies (1:400; Life Technologies, Grand Island, NY, http://www.invitrogen.com) were used to visualize incorporated BrdU. Cell nuclei were visualized by DAPI. Approximately 3,000 cells from control and TBX3 overexpressing hESC lines were quantified and normalized to the total cell number in each field using the Image J software.

Quantification of Neural Rosettes

Differentiated hESCs were fixed with 4% paraformaldehyde in PBS for 20 minutes, washed, and subsequently incubated with 10% blocking serum (10% goat serum and 0.1% Triton X-100 in PBS) containing DAPI (1:400) for 1 hour. DAPI images were taken of all neural rosette structures for control and TBX3 shRNA cell lines using a Nikon Eclipse Ti Inverted Microscope. Image J software was used to count total number of neural rosette structures. The total number of neural rosettes was then normalized to the number of EBs plated per cell line or the total number of cells at time of fixation. This experiment was performed in triplicate for control and TBX3 shRNA cell lines.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

TBX3 Is Upregulated in Differentiating hESCs

Although it has been shown that Tbx3 is necessary to maintain mouse ESC self-renewal and pluripotency, it is important to verify whether TBX3 exhibits a similar function in hESCs. In order to gain insight of TBX3's function in hESC pluripotency or differentiation, we first determined the expression pattern of TBX3 in undifferentiated and differentiated hESCs. hESCs were induced to differentiate through an EB intermediate for 5 days. On the fifth day, EBs were collected, plated, and then treated with various differentiating agents such as FBS (5%), BMP4 (100 ng/ml), and RA (1 μM) for an additional 11 days. hESCs began to differentiate, characterized by the loss of tight colony morphology (Fig. 1A). By day 11 of treatment with FBS, RA, BMP4, or withdrawal of bFGF, TBX3 expression was significantly upregulated (Fig. 1B). To determine the time course of TBX3 upregulation during differentiation, we induced formation of EBs for 7 days and induced differentiation for an additional 14 days with 10% FBS. We selected 10% FBS as our differentiating agent to maximize EB adherence. qRT-PCR analysis was performed at day 7 of EB formation and at days 4, 7, 11, and 14 of differentiation with 10% FBS. As shown in Figure 1C, qRT-PCR analysis revealed that TBX3 expression is upregulated during EB formation and as early as day 4 of FBS-induced differentiation. OCT4 expression level decreased with increasing time of differentiation, confirming that these cells are differentiating cells (Fig. 1C). At each time point of differentiation with 10% FBS, immunocytochemistry of TBX3 and OCT4 was performed and results were consistent with our qRT-PCR data. As shown in Figure 1D, TBX3 expression began to increase in differentiated cells as early as day 4 and continued increasing to day 11 (Fig. 1D). More importantly, cells that highly expressed TBX3 did not express OCT4. This was demonstrated at every time point during differentiation (Fig. 1D, insets). In contrast to mouse ESC studies in which Tbx3 expression is downregulated upon EB formation and differentiation with RA [12, 14, 25, 26], our results show that TBX3 is highly upregulated upon differentiation of hESCs. The reciprocal expression patterns of TBX3 and OCT4 imply that TBX3 may promote differentiation of hESCs by inhibiting OCT4 expression.

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Figure 1. TBX3 is upregulated upon differentiation of hESCs. (A): Cell morphologies of adhered EBs at day 11 of differentiation. Images were taken at ×4 magnification. (B): Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of relative TBX3 expression levels in differentiated hESCs at day 11 (undifferentiated H9 = 1.0). *, p <.05. TBX3 is upregulated approximately 45-fold, 30-fold, 35-fold, and 8-fold in H9 cells treated with FBS, withdrawal of bFGF, RA, or BMP4, respectively. (C): Quantitative RT-PCR analysis of relative TBX3 and OCT4 expression levels in EBs and 10% FBS induced differentiated hESCs at days 4, 7, 11, and 14 (undifferentiated H9 = 1.0). TBX3 is upregulated in EBs at day 7 and in 10% FBS induced differentiated hESCs at days 4, 7, 11, and 14, respectively. OCT4 expression levels decreased as time of differentiation increased. *, p <.05. (D): Immunocytochemistry of TBX3 and OCT4 expression in 10% FBS induced differentiated hESCs at days 4, 7, 11, and 14. Double staining for TBX3 (red) and OCT4 (green). TBX3 and OCT4 expression do not overlap at any time point (white arrow and insets). Images were captured at ×10 magnification. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; EB, embryoid body; FBS, fetal bovine serum; hESC, human embryonic stem cell; RA, retinoic acid; TBX3, T-box 3.

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TBX3 Does Not Regulate OCT4 Promoter Activity

As shown in Figure 1D, TBX3 expression did not overlap with OCT4 expression at any time during differentiation, suggesting that TBX3 may function to inhibit OCT4 expression and promote differentiation in hESCs. To test this, a hOCT4-eGFP hESC reporter line was created by transfection with the reporter construct, phOCT4-eGFP. To determine whether TBX3 inhibits OCT4 expression, these cells were transduced with lentivirus to overexpress TBX3. This construct has been used to generate stable hESC lines and exhibited similar regulation of OCT4p-eGFP expression to that of endogenous OCT4 in parental hESCs [24]. Figure 2A shows the hESC line expressing the eGFP reporter gene under the control of the OCT4 promoter. As shown in Figure 2B (arrows), TBX3 overexpression does not inhibit the expression of eGFP, suggesting that TBX3 does not directly regulate the OCT4 promoter. This data also suggest that the converse expression of TBX3 and OCT4 seen in differentiating hESCs, in Figure 1D, may represent two different stages of differentiation and that the absence of OCT4 in TBX3 positive cells is independent of the function of TBX3.

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Figure 2. TBX3 does not directly inhibit the human OCT4 promoter activity. (A): Human OCT4p-eGFP embryonic stem cell line. (B): Overexpression of TBX3 colocalizes with eGFP expression and does not inhibit the OCT4 promoter to decrease eGFP expression (white arrows). Images were taken at ×20 magnification. Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; eGFP, enhanced green fluorescence protein.

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Overexpression of TBX3 in Undifferentiated hESCs Promotes Stem Cell Proliferation but is Not Sufficient to Induce Differentiation

Since TBX3 is highly expressed during differentiation of hESCs, it is important to determine whether overexpression of TBX3, alone, is sufficient to initiate differentiation. In this experiment, we used lentivirus to generated stable hESCs lines overexpressing TBX3 tagged with Myc. Overexpression of TBX3 was verified by Western blot (Fig. 3A) and qRT-PCR (data not shown). For immunocytochemistry and Western blot analyses, anti-myc antibody was used to detect ectopically expressed TBX3 and to differentiate endogenous TBX3 (Fig. 3A, 3C). Three independent ESC lines overexpressing TBX3 (C5, C10, and C12) were used in subsequent experiments. Results of qRT-PCR analysis revealed no significant changes in the expression of pluripotency (OCT4, NANOG, and SOX2), endoderm (AFP), or mesoderm (HAND1) markers in all TBX3 overexpressing clones, except for PAX6 (ectoderm) which was significantly downregulated, and SOX1 (ectoderm) which was significantly upregulated (Fig. 3B). Confirming our qRT-PCR data, immunocytochemistry analysis of OCT4 and NANOG revealed no change in the expression of these pluripotent markers upon overexpression of TBX3 (Fig. 3C). A recent study suggested that overexpression of Tbx3 in mouse ESCs induced differentiation into ExEn by direct upregulation of Gata6 expression [14]. To test whether TBX3 plays the same role in hESCs, we examined GATA6 expression with qRT-PCR. Our result showed no change in GATA6 expression, suggesting overexpression alone is not sufficient to drive undifferentiated hESCs to a specific lineage. This data show that the overexpression of TBX3 alone is not sufficient to induce differentiation of undifferentiated hESCs, further supporting that TBX3 functions differently in human than in mouse ESCs.

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Figure 3. Overexpression of TBX3 in undifferentiated human embryonic stem cells (hESCs) does not affect pluripotency. (A): Verification of ectopically expressed myc-TBX3 in hESCs. TBX3 overexpression was verified by Western blot. (B): Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of pluripotency and differentiation markers in TBX3 overexpressing clones. Expression levels of pluripotency (OCT4, NANOG, and SOX2) and differentiation-related genes (AFP, HAND1, PAX6, and SOX1) were analyzed by qRT-PCR in TBX3 overexpressing clones (Control H9 cells = 1.0) *, p <.05. (C): Immunocytochemistry of TBX3, OCT4, and NANOG expression in control and TBX3 overexpressing hESC lines. TBX3 overexpressing cells maintained normal OCT4 (second row, white arrows) and NANOG (fourth row, white arrows) expression when compared with control cells (first and third rows). Images were taken at ×20 magnification. Abbreviation: TBX3, T-box 3.

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Although TBX3 overexpression did not induce differentiation, the colonies overexpressing TBX3 appeared more compact and needed to be passaged more often than control ESCs (Fig. 4A). As shown in Figure 4A, approximately 72 hours after passage, while control ESC colonies contained only a single layer of cells, TBX3 overexpressing colonies had multiple cell layers. To determine whether this difference was due to increased cell proliferation, the mitotic index was calculated. Using DAPI staining, total and mitotic cells were identified and counted (Fig. 4A, insets). TBX3 overexpressing cells had a significantly higher mitotic index suggesting that these cells were undergoing increased cell proliferation (Fig. 4B). We also performed a BrdU incorporation assay to further verify our mitotic index results. As shown in Figure 4C, hESCs overexpressing TBX3 had a significantly higher percentage of cells that incorporated BrdU, suggesting that TBX3 overexpression promotes hESC proliferation in the undifferentiated state.

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Figure 4. Overexpression of TBX3 increases stem cell proliferation possibly by repressing the expression of NFκBIB and p14ARF. (A): Phase images of control (left) and TBX3 overexpressing (right) human embryonic stem cell (hESC) colonies cultured on mouse embryonic fibroblasts. Examples of DAPI images used to calculate mitotic index. Mitotic cells are shown in insets. (B): TBX3 overexpressing ESCs have a significantly higher mitotic index than control ESCs. *, p <.05. (C): TBX3 overexpression increases the percentage of BrdU-positive ESCs cells. *, p <.05 (D): TBX3 overexpression in hESCs results in a significant decrease in the expression of cell cycle regulators, NFκBIB and p14ARF. *, p <.05. Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; TBX3, T-box 3.

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TBX3 has been shown to inhibit p14ARF expression to promote proliferation and immortalization of mouse embryo fibroblasts [7, 27–31]. We have recently shown that TBX3 directly binds to and inhibits the promoter activity of NFκBIB, an inhibitor of NFκB, to promote mammary epithelial cell proliferation [32]. Since the NFκB pathway is known to regulate cell cycle progression and proliferation [33, 34], we decided to examine NFκBIB and p14ARF expression levels. As shown in Figure 4D, both NFκBIB and p14ARF expression were significantly decreased upon overexpression of TBX3, suggesting that TBX3 overexpression may increase hESC proliferation by repressing NFκBIB and p14ARF expression.

Knockdown of TBX3 Impairs Neuroepithelial Differentiation in Differentiating hESCs

To further investigate the role of TBX3 during hESC differentiation processes, we established hESC lines that were stably integrated with shRNA targeting TBX3. Since undifferentiated hESCs express low levels of endogenous TBX3, we used a readily available MCF7-TBX3-eGFP cell line, expressing a high level of the TBX3-eGFP fusion protein, to test the efficiency of the TBX3 shRNA lentiviral constructs. Western blot analysis of MCF7-TBX3-eGFP cells transfected with lentiviral constructs, harboring shRNA targeted at various exons of TBX3, efficiently knocked down TBX3 expression (Fig. 5A). TBX3 level was not affected in cells transfected with the control vector or the 3′UTR shRNA vector, as expected (Fig. 5A). To create stable TBX3 shRNA hESC lines, a lentiviral vector harboring shRNA targeted at exon 2 of TBX3 was used to produce lentivirus and transduce hESCs. Two clones of control (Control-1 and -2) and TBX3 shRNA (TBX3shRNA-1 and -2) ESC lines were used for subsequent experiments.

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Figure 5. TBX3 knockdown inhibits neural rosette formation in differentiated human embryonic stem cells. (A): Verification of TBX3 knockdown. MCF7-TBX3-enhanced green fluorescence protein (eGFP) cell line was transfected with various TBX3 shRNA lentiviral vectors. Cell lysates were harvested and a Western blot was performed, using a rabbit anti-TBX3 antibody, to check the efficiency of TBX3 knockdown. TBX3-eGFP fusion protein runs at 97 kDa while myc-TBX3 protein runs at 72 kDa. (B): Phase images of cell morphologies at day 8 of differentiation with 10% fetal bovine serum suggest a decrease in the number of neural rosette structures. (C): Quantification of neural rosettes in control and TBX3 knockdown cell lines. The loss of TBX3 expression coincided with a significant loss in neural rosette formation.*, p <.05. (D): Characterization of neural rosettes. Immunocytochemistry was performed with antibodies for PAX6, SOX1, NESTIN, and ZO-1. Radial formation of NESTIN, apical localization of ZO-1, and localization of mitotic cells within the apical lumen can be seen within the insets. Images were taken at ×10 magnification. Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; TBX3, T-box 3.

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Previous studies in mouse ESCs have shown that knockdown of Tbx3 compromises self-renewal and results in differentiation, suggesting that Tbx3 is important for maintaining self-renewal [12–14]. However, our human TBX3shRNA ESC lines showed no obvious changes to colony morphology (Supporting Information Fig. S1A). Furthermore, qRT-PCR analysis of pluripotency markers, OCT4 and NANOG, showed no significant change in expression when compared to control cells (Supporting Information Fig. S1B). To examine whether the knockdown of TBX3 in undifferentiated hESCs affected stem cell proliferation, the mitotic index was calculated as previously described. Knockdown of TBX3 did not affect cell proliferation in undifferentiated hESCs (Supporting Information Fig. S1C). Since TBX3 expression in undifferentiated hESCs is extremely low, possibly due to TBX3 being epigenetically repressed by Sirtuin-1 (NAD+-dependent class III histone deacetylase) [35], further knockdown of TBX3 at this stage was not expected to affect pluripotency or cell proliferation.

To determine whether TBX3 plays a role in differentiating hESCs, TBX3 shRNA ESC lines were induced to form EBs and cultured in suspension for 7 days without bFGF. On the seventh day, EBs were plated and fed with hESC medium supplemented with 10% FBS for 8 additional days. Colony morphology was examined. As shown in Figure 5B, there was a decrease in the number of neural rosette-like structures in the TBX3 knockdown ESC line. To verify that these structures were indeed neural rosettes, we performed immunocytochemistry of PAX6, SOX1, NESTIN, and ZO-1 (Fig. 5D). PAX6 and SOX1 are early neural transcription factors and commonly used as markers of neural rosettes [36–41]. Besides the typical radial cell morphology seen by DAPI staining, the expression pattern of PAX6 and SOX1 is consistent with neural rosettes (Fig. 5D). NESTIN is a well-known neural cell marker that is expressed within rosettes [42, 43]. NESTIN expression with radial appearance is consistent with morphology of neural rosettes (Fig. 5D, inset). Formation of neural rosettes is initiated by acquiring cell polarity that is illustrated by the redistribution of ZO-1, a tight junction protein [44]. As shown in Figure 5D, ZO-1 expression was localized to the apical lumen of our structures as observed in neural rosettes [44]. Neural rosettes are also characterized by the presence of mitotic cells within the apical lumen [40]. Mitotic cells were identified in the apical lumen of these structures (Fig. 5D, DAPI insets). Together, expression of these markers confirms that the identified structures are neural rosettes. After characterization, neural rosettes were quantified and normalized to the number of EBs plated. The number of neural rosettes/EB plated significantly decreased upon knockdown of TBX3 in differentiating cells (Fig. 5C). qRT-PCR analysis revealed that knockdown of TBX3 upregulated mesoderm (BRACHYURY and HAND1) and endoderm markers (AFP and GATA4), suggesting that TBX3 may function to inhibit differentiation into mesoderm and endoderm lineages (Fig. 6A). Interestingly, the ectoderm marker, PAX6, was downregulated while SOX1 expression remained unchanged, suggesting that knockdown of TBX3 impairs differentiation into neuroepithelia (Fig. 6A, 6B). This result is consistent with the decrease in neural rosette formation. Expression of OCT4, NANOG, and SOX2 is upregulated in TBX3 knockdown cell lines (Fig. 6B), suggesting that TBX3 may function to enhance repression of these markers during differentiation.

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Figure 6. TBX3 knockdown inhibits neuroepithelial, neuroectoderm, and eye field marker expression. Expression levels of pluripotency and differentiation-related markers in TBX3 knockdown cell lines at day 8 of differentiation with 10% fetal bovine serum were analyzed. (A): Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on the expression of TBX3, endoderm markers (AFP and GATA4), mesoderm markers (BRACHYURY and HAND1), and neuroepithelial markers (PAX6 and SOX1). Control cells = 1.0. *, p <.05. (B): qRT-PCR was performed on the expression of TBX3, pluripotency markers (OCT4, NANOG, and SOX2), neuroepithelial markers (PAX6 and SOX1), forebrain markers (FOXG1 and LHX2), and eye field markers (PAX6, LHX2, and RAX). *, p <.05. Abbreviation: TBX3, T-box 3.

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To confirm that TBX3 knockdown specifically affects neuroepithelial differentiation, we differentiated both control and TBX3 shRNA hESC lines using a well-defined neuroepithelial differentiation protocol previously described by Shi et al. [23]. Briefly, control and TBX3 shRNA ESC lines were plated onto matrigel and once cells reached 90% confluence, neural induction was initiated by changing the culture medium to one that supports neural induction, referred to as 3N medium [23], for an additional 14 days. The efficiency of neural induction between control and TBX3 shRNA cell lines was monitored by the appearance of cells with characteristic neuroepithelial cell morphology (neural rosette formation). After characterization, neural rosettes were quantified and normalized to the number of cells present at day 14. Using this defined neuroepithelial differentiation protocol, we have found that knockdown of TBX3 results in a significant decrease in neural rosette formation, as compared to control cells (Supporting Information Fig. S1D). This result is consistent with our differentiation protocol and suggests that TBX3 plays an important role in early neuroepithelial differentiation.

Tbx3's role in Xenopus eye field formation has been well-characterized [45]. Eye field transcription factors (pax6, rx1, tbx3/ET, six3, lhx2, and six6) are considered important for eye field formation [45]. Overexpression of these factors in the developing Xenopus embryos can induce eye-like structures [46]. Moreover, BMP4 regulates Tbx3 expression in the developing optic cup of mice eyes [47, 48]. TBX3 was found to be expressed in the ciliary epithelium of the adult mammalian eye [49]. To test whether knockdown of TBX3 affected eye field specification, we performed qRT-PCR analysis on the expression of eye field markers (PAX6, LHX2, and RAX). Since commitment toward a retinal lineage begins with the establishment of the eye field within the anterior neuroepithelium [32], we also performed qRT-PCR on anterior/forebrain markers (LHX2 and FOXG1). Interestingly, the expression of both anterior/forebrain and eye field markers was significantly reduced in TBX3 knockdown differentiated cells (Fig. 6B). These results suggest that TBX3 may not only function to promote neuroepithelial differentiation but also function to promote eye field specification.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In this study, we have defined new roles for TBX3 in hESCs. We found that TBX3 overexpression, in the undifferentiated state, promotes increased stem cell proliferation possibly through the repression of NFκBIB and p14ARF (Figure 7). Knockdown experiments, during differentiation, revealed an unrecognized role of TBX3 in neuroepithelial differentiation (Figure 7). To gain insight into whether TBX3 plays a role in promoting hESCs self-renewal or differentiation, we examined TBX3 expression during differentiation of hESCs. To our surprise, we found TBX3 expression to be significantly upregulated as early as EB formation and further upregulated during differentiation with BMP4, FBS, and RA, suggesting a possible role of TBX3 in promoting hESCs differentiation. This result is completely opposite of what has been shown in mouse ESCs. In mouse ESCs, Tbx3 is highly expressed in the undifferentiated state and is downregulated upon EB formation and differentiation with RA or PI3K inhibitor [12, 14]. ChIP sequencing has shown that Tbx3 binds to the Oct4 promoter in mouse ESCs [15] to promote self-renewal and pluripotency. Since hESCs and mouse ESCs represent different stages of pluripotency, our study suggests that they may also have distinct responses to the manipulation of TBX3. Examples in which hESCs and mouse ESCs respond differently to similar transcriptional and signaling pathways have been well-documented. Addition of LIF or BMPs to mouse ESCs is necessary to maintain pluripotency while these same factors cause differentiation in hESCs [18–22].

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Figure 7. Model for TBX3 regulation of human ESC (hESC) proliferation and differentiation. In undifferentiated hESCs, TBX3 may promote cell proliferation by repressing cell cycle regulators, NFκBIB and p14ARF. During differentiation, TBX3 may function to promote neuroepithelial differentiation while inhibiting differentiation into mesoderm and endoderm. Abbreviations: EB, embryoid body; ESC, embryonic stem cell.

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Although hESC lines overexpressing TBX3 had no effect on the expression of pluripotency markers (OCT4, NANOG, and SOX2), we found that ectoderm marker PAX6 was significantly downregulated while SOX1 was significantly upregulated. Currently, there are no studies suggesting that TBX3 directly regulates PAX6 or SOX1 in hESCs. However, a study by Pankratz et al. showed PAX6 to be downregulated and SOX1 to be upregulated by day 6 of EB formation when compared with undifferentiated hESCs [41]. The similar PAX6 and SOX1 expression pattern between these two studies invites further investigation into the relationship between PAX6, SOX1, and TBX3 in hESCs. Overexpression of Tbx3 was found to promote mouse ESC differentiation into ExEn by direct upregulation of Gata6 expression. In our study, TBX3 overexpression did not affect GATA6 expression to induce differentiation, confirming that TBX3 plays different roles in hESCs and mouse ESCs.

TBX3 overexpressing clones had a significantly higher mitotic index and BrdU incorporation than control cells and both p14ARF and NFκBIB were significantly repressed. These results are consistent with TBX3 function, identified by our and other groups, to promote cell proliferation possibly by repressing p14ARF [28, 29, 50, 51]. ARF transcript levels are repressed in iPS and ESCs compared to MEFs [52]. In hematopoietic and neural stem cells, Bmi-1 was found to support self-renewal by suppressing the expression of p19/p14ARF [53–55], suggesting that p14/p19ARF inhibits proliferation for various types of stem cells. Our study suggests that by inhibiting p14ARF, TBX3 promotes hESC proliferation. NFκBIB, an inhibitor of NFκB, is also repressed by TBX3. NF-κB associated pathways also play an important role in cell proliferation, differentiation, and apoptosis [35]. Aberrant activation of NFκB results in excessive cellular proliferation [56]. Our results are consistent with our recent study which showed that TBX3 overexpression directly inhibits NFκBIB expression to promote mammary epithelial proliferation [50]. However, NFκB signaling in hESCs is controversial [57, 58]. In this study, we showed that TBX3 inhibited NFκBIB expression, suggesting additional pathways that promote hESC proliferation. Further studies assessing the expression of targets of the p14ARF and NFκB signaling pathway will further elucidate the mechanism by which TBX3 expression regulates hESC proliferation.

Our study showed that knockdown of TBX3 in the undifferentiated state of hESCs did not attenuate self-renewal or pluripotency to induce differentiation. This is consistent with previous findings in which TBX3 expression is expected to be extremely low in the undifferentiated state [35] and thus further reducing the level of TBX3, at this stage, would not affect self-renewal or pluripotency. However, TBX3 knockdown during differentiation of hESCs resulted in an upregulation of OCT4, NANOG, and SOX2. Since our overexpression studies showed that TBX3 does not directly regulate the expression of OCT4 and NANOG, TBX3 may influence differentiation through another mechanism. BMP signaling promotes hESC differentiation. TBX3 has been identified both upstream and downstream of BMP signaling [59, 60]. Additional studies to determine whether TBX3 regulates the BMP signaling pathway to enhance hESC differentiation will facilitate our understanding of these mechanisms. Knockdown of TBX3 resulted in a significant decrease in neural rosette formation, suggesting a role for TBX3 in promoting neuroepithelial differentiation. This was confirmed by a decrease in PAX6 expression. However, the expression of another neuroepithelial marker, SOX1, did not change. Studies have shown that during neural differentiation of hESCs, initial neuroepithelial cells express PAX6 much earlier than SOX1 [38, 41]. PAX6 expression was detected as early as day 6 of differentiation while SOX1 expression was detected at day 14. Thus, change of SOX1 expression in our TBX3 knockdown cells may not have been initiated yet. If we extended our time point to allow further differentiation, we may be able to better assess if knockdown of TBX3 also affects SOX1 expression. In these TBX3 shRNA differentiation experiments, we used a nondefined hESC culture system with MEF and FBS. It is important to note that this differentiation method had certain limitations, especially since nondefined factors from MEF and FBS could influence differentiation toward one specific lineage over another. Thus, the direct effect of TBX3 knockdown on neuroepithelial differentiation could be skewed by nonspecific effects based on our initial differentiation method. To eliminate this possible effect, and examine the effect of TBX3 knockdown on neuroepithelial differentiation, we repeated our TBX3 shRNA differentiation experiment using a previously published and well-defined neuroepithelial differentiation protocol, as described by Shi et al. Using this protocol, we have recapitulated our previous results in which TBX3 knockdown results in a decrease in neural rosette formation, further suggesting that the lack of TBX3 specifically affects differentiation toward neuroepithelial cells.

It is very interesting that knockdown of TBX3 impaired neural rosette formation and decreased the expression of anterior/forebrain (FOXG1 and LHX2) and eye field markers (LHX2, PAX6, and RAX), further suggesting a role for TBX3 in regulating neuroepithelial differentiation. This result is consistent with findings in Xenopus in which Tbx3 regulates eye field specification [45, 46]. To confirm TBX3's role in regulating differentiation toward neuroepithelia is not due to the aberrant differentiation of other germ layers during EB formation, additional experiments with lineage-specific differentiation protocols will clarify the function of TBX3 in eye field and neuroepithelial development.

This study demonstrates that TBX3 functions differently in hESCs. In mouse ESCs, Tbx3 expression is necessary for maintaining self-renewal, while overexpression promotes differentiation into ExEn [12–14]. In undifferentiated hESCs, TBX3 promotes ESC proliferation possibly through the repression of NFκBIB and p14ARF (Figure 7). During differentiation, TBX3 may promote neuroepithelial and eye field specification (Figure 7). hESCs and mouse ESCs represent distinct pluripotent states which may explain why TBX3 functions differently in hESCs than it does in mouse ESCS. hESCs are more similar to mouse-derived epiblast stem cells than mouse ESCs [17, 61, 62], suggesting that hESCs are at a later developmental stage that is more primed for differentiation. Due to their different pluripotent and developmental states, hESCs and mouse ESCs may respond to TBX3 manipulation differently.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Our results suggest TBX3 promotes hESC proliferation in the undifferentiated state and plays a role to promote neuroepithelial differentiation. These results provide new insight into the roles of TBX3 in regulating self-renewal and differentiation of hESCs. Further investigation of the molecular mechanisms by which TBX3 regulates neuroepithelial differentiation will contribute to our understanding of ESC differentiation and specification in early human development.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank Chengkang Zhang for cloning vectors used in this study. We would also like to thank the UCI Stem Cell Core Facility for providing training and cell culture space. The authors are grateful to the Huang Lab for critically reading this manuscript. This work was supported by the National Cancer Institute R01CA121876 Grant to T.H. and R01CA121876 minority supplemental to T.E.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
SC-12-0123_sm_SupplFigure1.tif1749KFigure S1. Knockdown of TBX3 does not affect human ESC pluripotency or cell proliferation. (A) Knockdown of TBX3 in human ESCs did not affect colony morphology. (B) Quantitative RT-PCR analysis of pluripotency markers, OCT4 and NANOG. (Control H9 cells =1.0) TBX3 knockdown did not significantly affect OCT4 or NANOG expression. (C) Knockdown of TBX3 did not affect cell proliferation. Mitotic index was calculated as described previously for the over-expression studies. (D) Quantification of neural rosettes in control and TBX3 knockdown cell lines using a defined neuroepithelial differentiation protocol. The loss of TBX3 expression coincided with a significant loss in neural rosette formation.*, p<0.05.

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