Activin A Maintains Self-Renewal and Regulates Fibroblast Growth Factor, Wnt, an - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:05 |只看该作者 |倒序浏览 |打印

作者：Lei Xiao, Xuan Yuan, Saul J. Sharkis作者单位：Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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      【摘要】: L, S1 m4 ?7 ~6 }- I/ ^
   Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.
      【关键词】 Embryonic stem cell totipotency: D* t" v  H* u1 k7 v( q& O/ C4 B
   INTRODUCTION
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Human embryonic stem cells (hESCs) are derived from the blastocyst. These cells are capable of unlimited proliferation and maintain pluripotency in vitro . Thus, hESCs provide potential applications in regenerative medicine and study of early human development. However, little is known about the regulatory mechanisms that maintain hESC self-renewal and pluripotency.; a( u" p. I; y& g  Q
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Two intrinsic transcriptional factors, Oct4 (also known as Pou5f1) and Nanog, are required for the maintenance of the undifferentiated state of embryonic stem (ES) cells . Thus, several factors play regulatory roles in the ESCs of mouse, human, or both.* \3 Z- e' n6 N
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Nodal/Activin signals establish the embryonic axes, induce mesoderm and endoderm, pattern the nervous system, and determine left-right asymmetry in vertebrates .
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Here we report that Activin A functions to maintain hESC self-renewal and pluripotency. We observed that upregulation of Nodal/Activin, FGF, Wnt, and Hedgehog (Hh) signals and downregulation of BMP and Notch signals are correlated with increased expression of Oct4 and Nanog in an aneuploid hESC line (referred to as H1T; T stands for translocation). Activin A, unlike Nodal, was necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and was able to support long-term feeder-free growth of hESCs. Importantly, we demonstrated that Activin A was able to induce the expression of other regulators, such as Oct4, Nanog, Nodal, Wnt3, bFGF, and FGF8 and slightly suppressed the expression of BMP7. Therefore, our data revealed a central role of Activin A in maintaining hESCs stemness.( B" h( v) O$ ^9 }- F
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MATERIALS AND METHODS

hESC Culture
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hESC line H1 was obtained from WiCell Research Institute (Madison, WI, http://www.wicell.org), and I-6 was obtained from Technion-Israel Institute of Technology (Haifa, Israel, http://www.technion.ac.il). hESCs were maintained on feeders in hESC medium, which contains 80% Dulbecco¡¯s modified Eagle¡¯s medium (DMEM)/Ham¡¯s F-12 medium (F12) medium, 20% knockout serum replacement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids, and 4 ng/ml human bFGF. H1 cells were passaged approximately once a week by incubation in 1 mg/ml collagenase IV for ~30 minutes at 37¡ãC. H1T cells were passaged approximately once a week by incubation in 0.25% Trypsin for ~5 minutes at 37¡ãC. Conditioned medium (CM) was collected according to Xu et al. . Non-CM contains 80% DMEM/F12 medium, 20% knockout serum replacement (Invitrogen), 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids. For long-term feeder-free culture, 5 ng/ml Activin A was added in non-CM. Recombinant human Activin A, mouse Nodal, human Lefty-A, and human Follistatin were purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com).
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Karyotype Analysis* z! u+ [" _0 U0 r

Karyotype analysis (G-banding) was performed on at least 20 cells from each sample. Multicolor spectral karyotyping (SKY)  was performed according to the protocol supplied with the probes (Applied Spectral Imaging, Carlsbad, CA).

Analysis of Cloning Efficiency
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The cloning efficiency was determined as described by Amit et al. .3 J/ l3 _6 _, O+ T$ L) y

Flow Cytometry& f8 f2 X0 F) t. p  d) x! n) }
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Dissociated hESCs were suspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) at a concentration of 1 x 106 cells per 100 µl and add SSEA-4 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww). After 30 minutes at 4¡ãC, the cells were washed once and resuspended in PBS containing 1% BSA. Then fluorescein isothiocyanate-conjugated goat anti-mouse IgG was added. Finally, the cells were washed twice and stained with propidium iodide. Live cells identified by propidium iodide exclusion were analyzed for surface marker expression using FACSCalibur (BD Biosciences, San Diego, http://www.bdbiosciences.com) and Cell Quest software (BD Biosciences).1 E( k- p5 s# K% _0 N  l0 R
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Immunostaining  |( ?& q/ D( t3 R

Immunostaining was carried out similarly as described . Antibodies used were anti-Oct4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-Nanog (R&D Systems), anti-SSEA3 (Developmental Studies Hybridoma Bank), anti-SSEA4 (Developmental Studies Hybridoma Bank), anti-tra-1¨C60 (Chemicon, Temecula, CA, http://www.chemicon.com), anti-tra-1¨C81 (Chemicon), anti-pan-cytokeratin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), anti-smooth muscle actin (Sigma-Aldrich), anti--fetoprotein (Sigma-Aldrich). For Oct4 and Nanog immunostaining, cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes followed by permeabilization for 20 minutes in 100% ethanol.

Teratoma Formation; O  I/ f6 K7 t2 I6 `( i

Cells were injected intramuscularly into non-obese diabetic/severe combined immune deficient (NOD/SCID) mice (~5 x 106 cells per site). Four mice were injected for each cell line; all of the mice formed teratomas. After 4¨C6 weeks, tumors were processed for hematoxylineosin staining similarly as described . All animal experiments were conducted in accordance with the Guide for the Care and Use of Animals for Research Purposes and were approved by the Johns Hopkins Animal Care Committee.7 t3 B- Y6 B5 \3 P7 U1 e' K
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Microarray! y' `5 {" S8 G  R4 q; N6 |

Total RNA was extracted with TRIZOL reagent (Invitrogen) and further purified using an RNeasy column (Qiagen, Hilden, Germany, http://www1.qiagen.com). The labeling procedure was carried out by using a RNA Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). Fragmentation was carried out by incubating at 60¡ãC for 30 minutes in fragmentation buffer (Agilent Technologies) and stopped by adding equal volume of 2x hybridization buffer (Agilent Technologies). Fragmented target was applied to a Whole Human Genome Oligo Microarray that contains 41,000 genes (Agilent Technologies). Hybridization proceeded at 60¡ãC for 17 hours in a hybridization oven (Robbins Scientific, Sunnyvale, CA, http://www.robsci.com). The hybridized array was scanned with Agilent G2565BA microarray scanner. The TIFF image generated was loaded into Feature Extraction Software (Agilent Technologies) for feature data extraction.7 ^- s$ n+ F, p5 d
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Immunoblotting
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Cells were lysed with 1x lysis buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, and complete mini protease inhibitor cocktail (Roche Diagnostics, Basel Switzerland, http://www.roche-applied-science.com). Total protein (10 µg) was loaded for each lane. Membranes were blocked in Tris-buffered saline with 0.1% Tween and 5% milk. Antibodies used were anti-phospho-Smad2/3 (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), anti-Smad2/3 (Cell Signaling), anti-Oct4 (Santa Cruz Biotechnology), anti-human Nanog (R&D Systems), and ß-Actin (Abcam, Cambridge, MA, http://www.abcam.com). Primary antibodies were incubated overnight, and secondary antibodies were incubated for 2 hours. Proteins were detected by chemiluminescence (Pierce, Rockford, IL, http://www.piercenet.com).
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TRAP Assay1 v  I, }& m1 W% H
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Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) assay as described ., C4 W: C8 S' U

RNA Isolation and Real-Time Reverse Transcription-Polymerase Chain Reaction$ ^2 j) `+ S/ y+ ^4 p
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Total RNA was prepared using the RNAeasy kit (Qiagen) and used as a template for reverse transcription-polymerase chain reaction (RT-PCR). Real-time PCR was performed in MyiQ real-time PCR detection system (Bio-Rad, Hercules, CA, http://www.bio-rad.com) using an Synergy Brand GreenI-based PCR Master mix (Bio-Rad). PCR primers are listed in the supplemental online Table 1. Each experiment was carried out at least three times. The expression value of each gene was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase cDNA to calculate a relative amount of RNA present in each sample. The expression level of each gene in non-CM was arbitrarily defined as 1 unit. The normalized expression values for all control and treated samples were averaged, and an average fold change was determined. Analysis of variance (ANOVA) was conducted between the normalized relative expression values for control and treated samples to determine statistical significance.! c. n( H: ~; U: x" E1 Y. `

Table 1. Gene expression analysis of signaling pathway genes in H1T versus H1 by microarray and RT-PCR" d# v6 s. y7 t9 ]) [

RESULTS- E# L) C# ]+ c! z+ D* H# O

Upregulation of Activin A Correlates with Increased Expression of Oct4 and Nanog4 A3 ?  a3 a' E8 b% Y# z$ w( N

As has been reported by others , we detected aneuploidy in the H1 hESC line in our laboratory, after long-term passage with trypsin. The chromosomal translocation included a net gain of the long arm of chromosome 17 and a balanced (reciprocal) translocation between the 10th and 17th chromosome, without apparent loss of chromosome 10 material (Fig. 1A; data not shown). We refer to this line as the H1T hESC line.0 |1 ~: K8 A" {
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Figure 1. Aneuploid H1T cells obtained growth advantage without loss of pluripotency. (A): H1T human embryonic stem cells (hESCs) hold an extra copy of 17q and a translocation from chromosome 10¨C17 without obvious loss of chromosome 10 material. (B): Both H1 and H1T express undifferentiated hESC markers Oct4, Nanog, SSEA3, SSEA4, Tra-1¨C60, and Tra-1¨C81. (C): Both H1 and H1T cells are able to form complex teratomas including three germ layers: APC-positive epidermal tissue (ectoderm), smooth muscle actin-expressed muscle tissue (mesoderm), and AFP-positive liver tissue (endoderm). Scale bars = 25 µm. Abbreviations: AFP, -fetoprotein; APC, anti-pan-cytokeratin; SMA, smooth muscle actin.
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The cloning efficiency of euploid H1 hESCs was 0.22% ¡À 0.03% (n = 3). H1T cells showed a marked increased cloning efficiency of 8.62% ¡À 0.13% (n = 4). Thus, H1T cells gained a proliferative advantage evidenced by approximately a 40-fold increase in cloning efficiency compared with that of the H1 parent cell line (p 8 C) f$ W3 z) [( y. E
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The self-renewal advantage is a desirable feature of hESCs. Therefore, the aneuploid H1T hESCs provide a valuable tool to query the mechanism underlying self-renewal of hESCs. We assumed that if we discovered differences between H1T and H1 cells that conferred a self-renewal advantage on the H1T cells, we would aid in identifying the mechanism(s) that regulated the self-renewal of hESCs. Based on this assumption, we performed a microarray analysis comparing the gene expression profile of H1T hESCs and H1 hESCs.3 E1 h( I# Y& S2 v: y/ y

The microarray analysis was performed according to the manufacturer¡¯s instructions (Agilent Technologies). Fold change and p value were processed with Feature Extraction Software (Agilent Technologies). We observed that the H1T cells expressed 50% more Oct4 than H1 cells did (Table 1). It has been reported that a precise level of Oct4 is required to maintain the undifferentiated state of mESCs. The threshold for inducing differentiation is apparently set at 50% above or below the normal diploid expression level of Oct4 in undifferentiated mESCs . Therefore, a 50% change in Oct4 expression in H1T cells would be biologically significant. We defined the significantly changed genes as the genes that showed a fold change 1.5 and p value .05. According to these criteria, there were 830 significantly upregulated genes and 1,254 down-regulated genes in H1T cells versus H1 cells. Selected genes differentially expressed and associated with the major developmental signal pathways and/or pluripotency are shown in Table 1. We used real-time PCR to confirm the expression change of some key regulators (Table 1).
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The expression of ESC hallmarks, Oct4 and Nanog, showed increases of 1.50- ¡À 0.16-fold and 1.98- ¡À 0.19-fold in H1T hESCs versus H1 hESCs, respectively (Table 1)./ |, [3 r; @$ q2 b: ]
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The Wnt signal was upregulated in H1T cells, as evidenced by the increased expression of a ligand, Wnt3, and decreased expression of two inhibitors of Wnt signaling, SFRP1 and FRZB (Table 1). Downregulation of receptors (FZD2 and FZD8) and upregulation of the inhibitor (WIF) might be due to feedback inhibition  (Table 1).
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The expression of FGF ligands, bFGF and FGF8, increased in H1T hESCs, whereas the expression of FGF receptors 1, 2, and 3 decreased. SPRY4, an inhibitor of the FGF pathway, was upregulated (Table 1). These indicated that FGF signaling was upregulated and that a feedback inhibition mechanism could be involved.# h6 j! N7 {1 P. ?6 A
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Nodal was upregulated 3.1- ¡À 0.48-fold. Lefty-A and Lefty-B are the downstream targets and feedback inhibitors of Nodal signaling . They showed increases in the H1T cell line. Activin A increased 1.51- ¡À 0.01-fold, and a potential inhibitor of Activin A, Follistatin-like 1, was downregulated in H1T hESCs (Table 1). These data indicated that Nodal/Activin signals were upregulated. Activin A receptor type IIB decreased 1.64- ¡À 0.13-fold, perhaps once again due to feedback inhibition.

BMP signal was downregulated in H1T hESCs, as evidenced by reduced expression of BMP2, BMP7, and BMP11 (Table 1). Notch signaling was also downregulated, as indicated by the reduced expression of ligands JAG1, DLK1, and DLL1 and downstream effect factor HES1 (Table 1).
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We did not detect a significant change in expression of the ligands for the Hedgehog pathway (data not shown). Patched is a receptor and an inhibitor of Hh pathway . The downregulation of Gli3 also suggested that Hh signaling might be upregulated (Table 1).

We also observed downregulation of differentiation markers for all three germ layers, including neurofilament (marker of ectoderm), cardiac muscle actin (mesoderm), and -fetoprotein (marker of endoderm) (supplemental online Table 2). There were no trophoblast genes that showed significant change in the H1T cells versus H1 cells. Combined with the evidence that H1T cells expressed hESC markers (Fig. 1B) and maintain pluripotency (Fig. 1C), these observations indicated the absence of differentiation in H1T cells.+ k* w2 Y. T3 X1 t' L! P
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In summary, the gene expression data by microarray analysis suggest that the transcription factors associated with pluripotency (Oct4 and Nanog, and Nodal/Activin, Wnt, FGF, and Hedgehog signaling pathways) were upregulated, whereas BMP and Notch pathways were downregulated in H1T hESCs (Table 1). This observation suggested that Nodal/Activin, Wnt, FGF, and Hedgehog pathways positively regulate the expression of Oct4 and Nanog and contribute to maintaining hESCs in an undifferentiated state, whereas BMP and Notch signaling negatively regulate the expression of Oct4 and Nanog and contribute to differentiation.8 }" ~* m; b2 z, k

The Wnt/ß-catenin pathway has been reported to transiently maintain pluripotency of hESCs . The function of Nodal/Activin in hESCs has not been definitely determined. Our observations about differential expression of the gene products associated with Wnt, FGF, and BMP pathways are consistent with the existing literature. We hypothesized that the Nodal/Activin pathway might function to maintain the expression of Oct4, Nanog, and the stemness of hESCs.  T: d/ q$ P. }9 X

Activin A Is Necessary and Sufficient for Self-Renewal and Pluripotency of hESCs
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Lefty-A blocks Nodal signaling by a dual mechanism, it binds Nodal directly, and it also binds epidermal growth factor-Cripto-FRL-Cryptic domain co-receptors, such as TDGF-1 (also known as Cripto), thus preventing the assembly of an active Nodal/receptor complex . To investigate the function of Nodal in hESCs, we used Lefty-A to block the function of Nodal in H1 hESCs. When Nodal signaling was blocked by adding Lefty-A in the CM, the expression of Oct4 and Nanog was downregulated in a dose-dependent manner (Fig. 2A). This indicates that Nodal does contribute to maintaining the expression of Oct4 and Nanog.

Figure 2. Activin A is necessary and sufficient to maintain the expression of Oct4 and Nanog. (A¨CC): Western blot analysis of H1 human embryonic stem cells (hESCs) cultured for 6 days under various conditions as labeled. Membranes were probed with antibodies specific for p-smad2, Smad2, Oct4, Nanog, and ß-Actin (as a loading control). H1 (D¨CG) or I6 (H¨CI) hESCs were cultured for 6 days in CM (D), in non-CM (E, I), in non-CM supplemented with 10 ng/ml Activin A (F, H), and in CM supplemented with 300 ng/ml Follistatin (G). Cells were stained with an antibody specific for Oct4 (D¨CI). (E', G', I') show counterstaining with DAPI for (E, G, I). Scale bars = 50 µm. Abbreviations: ActA, Activin A; CM, conditioned medium; FST, Follistatin; p-Smad2, phosphorylated Smad2.. W, ?+ C" v- b/ m

Although Nodal is necessary to maintain the expression of Oct4 and Nanog, Nodal is not sufficient to fully maintain the expression of Oct4 and Nanog even at a concentration of 1,000 ng/ml (Fig. 2A).

Follistatin is an inhibitor of Activin A that functions by directly binding with this protein . Follistatin was able to inhibit the expression of Oct4 and Nanog in a dose-dependent manner under the specific conditions used (Fig. 2B, 2G), and the inhibition was reversed by adding Activin A (Fig. 2B). This indicates that Activin A is necessary to maintain the expression of Oct4 and Nanog. Activin A was able to fully maintain the expression of Oct4 and Nanog in a dose-dependent manner (Fig. 2C, 2F). We also used flow cytometry to detect a phenotypic marker of undifferentiated hESC, Tra-1¨C60, to evaluate the function of Activin A. We found that Activin A was able to significantly increase the number of Tra-1¨C60 expressing cells in a dose-dependent manner (supplemental online Fig. 1). The effect of Activin A on Oct4 and Nanog was reversed by adding Follistatin (Fig. 2C), indicating the specific action of Activin A on maintenance of Oct4 and Nanog. Activin A also maintained the expression of Oct4 in another hESC line, I-6 (Fig. 2H, 2I; supplemental online Fig. 3A, 3B).5 j! J9 N6 p5 l- Y( \$ D# B
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Figure 3. Activin A is able to support long-term feeder-free culture of H1 human embryonic stem cells (hESCs). H1 hESCs maintained in 5 ng/ml Activin A for 15 passages express undifferentiated hESC markers: Oct4 (A), Nanog (B), SSEA3 (C), SSEA4 (D), Tra-1¨C60 (E), Tra-1¨C81 (F). Scale bars, 50 µm. (G): The expression of Oct4 and Nanog in H1 hESCs cultured in Activin A analyzed by Western blotting at passages 9 and 15 showed stable maintenance of these two genes. (H): Telomeric repeat amplification protocol analysis of telomerase activity in H1 cells maintained in Activin A at passages 9 and 15. Five thousand cells were used for each sample. Telomerase-positive cells (  cells) were used as a positive control, and heat-inactivated (HI) samples were used as negative controls. Abbreviations: CM, conditioned medium; MEF, mouse embryonic fibroblast.

Activin A Supports Long-Term Feeder-Free Culture of hESCs+ G* a. H! O4 ~
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To determine the concentration of Activin A that best supports hESC growth without feeder cells, we used different concentrations of Activin A in hESC medium without bFGF. The medium containing 5 ng/ml Activin A was sufficient to maintain hESCs in an undifferentiated state on Matrigel-coated flasks without either feeder cells or conditioned medium (Fig. 3). H1 hESCs cultured in 5 ng/ml Activin A in Matrigel-coated flasks showed an appearance similar to that of the cells growing in CM. After cultured in Activin A for 15 passages, H1 cells were found to be still strongly positive for hESC markers Oct4, Nanog, SSEA3, SSEA4, Tra-1¨C60, and Tra-1¨C81 (Fig. 3A¨C3F). Oct4 and Nanog were stably maintained during prolonged culture in Activin A (Fig. 3G). The markers of differentiation were stably inhibited by activin A (supplemental online Fig. 2). H1 cells maintained in Activin A showed high levels of telomerase activity (Fig. 3H). To examine the potential for in vivo differentiation of the hESCs, we injected the H1 hESCs maintained in Activin A for 10 passages into NOD/SCID mice to test their ability to form teratomas. As cells maintained on feeder cells, cells cultured in Activin A generated complex teratomas with various differentiated tissue types comprising all three germ layers (Fig. 4; data not shown). H1 hESCs have been cultured for more than 150 days and >20 passages in feeder-free conditions supplied with 5 ng/ml Activin A in non-CM. Karyotype analysis by G-banding was carried out. The cells cultured with Activin A demonstrated a normal karyotype (supplemental online Fig. 4).+ I. w6 _2 c1 j8 `5 U, R  E- n; x

Figure 4. Human embryonic stem cells cultured in Activin A maintained pluripotency. H1 cells maintained in 5 ng/ml Activin A without feeder or conditioned medium for 10 passages were able to form teratomas. Neural tissue (A), bone (B), and liver cells (C) were identified in teratomas by hematoxylineosin staining. Pan-cytokeratin-expressed epidermal tissue (ectoderm) (D), smooth muscle actin-expressed muscle tissue (mesoderm) (E), and -fetoprotein-expressed liver tissue (endoderm) (F) were identified by immunohistochemistry staining. Scale bars = 100 µm.3 D9 o/ B$ ~1 m
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Activin A Regulates Wnt, FGF, Nodal/Activin, and BMP Signal Pathways
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Wnt, FGF, Nodal/Activin and BMP pathways were shown to form a complex signaling network to maintain the properties of hESCs (Table 1) . Upon withdrawal of CM, the expression of Activin A itself was upregulated fivefold in non-CM, suggesting that inhibitory factors for Activin A expression could exist in CM (Fig. 5J). Interestingly, Activin A suppressed the expression of itself in a concentration-dependent manner (Fig. 5J p,  .05, ANOVA). The expression of BMP7 was suppressed significantly by Activin A (Fig. 5L p,

Figure 5. Real-time polymerase chain reaction analysis of gene expression. H1 human embryonic stem cells were maintained in CM or non-CM supplemented with different concentration of Activin A for 6 days. The expression level of each gene in non-CM is arbitrarily defined as 1 unit. Abbreviations: bFGF, basic fibroblast growth factor; CM, conditioned medium; FGF, fibroblast growth factor.
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Figure 6. (A): Activin A inhibited differentiation caused by BMP4 in human embryonic stem cells (hESCs). H1 hESCs were maintained in CM or non-CM supplemented with different concentration of BMP4 with or without Activin A for 6 days. The expression of Oct4 and Nanog was analyzed by real-time polymerase chain reaction (PCR). (B): Activin A is able to maintain the expression of Oct4 and Nanog at a 10-fold lower concentration than bFGF. H1 hESCs were maintained in CM or non-CM supplemented with different concentration of Activin A or bFGF for 6 days. The expression of Oct4 and Nanog was analyzed by real-time PCR. Abbreviations: 10B, 10 ng/ml BMP4; 10B 1A, 10 ng/ml BMP4 1 ng/ml Activin A; 10B  10A, 10 ng/ml BMP4 10 ng/ml Activin A; 10B  100A, 10 ng/ml BMP4 100 ng/ml Activin A; bFGF, basic fibroblast growth factor; CM, conditioned medium.3 K7 g1 d2 v; ^% T6 s, L3 ?
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bFGF has been shown to be sufficient to maintain the pluripotency of hESCs during long-term feeder-free culture with or without the addition of Noggin, a potent BMP signal inhibitor . We decided to compare the activity of bFGF and Activin A in terms of maintaining the expression of Oct4 and Nanog. We observed a 10-fold reduction in the ability of bFGF compared with Activin A in maintaining Oct4 and Nanog expression (Fig. 6B).! c; \+ K# P, n7 R5 a: W, I
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Therefore, our data suggest a complex regulatory network that maintains the properties of hESCs. In this network, Nodal/Activin, FGF, and Wnt positively contribute to the maintenance of the undifferentiated state of hESCs. The BMP pathway is implicated in negatively regulating the maintenance of the hESC properties. Activin A is able to maintain the expression of Oct4 and Nanog in hESCs, at lower concentrations than bFGF.! G% N& r! g; n
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DISCUSSION) S* Z* ]( w( R3 b% s& N
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Oct4 is an octamer motif binding homeodomain transcription factor. ES cell lines express high levels of Oct4, and precise levels of this gene are required to maintain the ES cell undifferentiated state .5 V4 z1 N- m) b# U; p2 S, i

It was reported that an increased dosage of chromosome 17q provides a selective growth advantage for the propagation of undifferentiated hESCs . These observations imply that selection pressure exists within the current hESC culture system, which favors the aneuploid hESC population, with enhanced proliferative ability conferred by increased expression of Oct4 and Nanog.5 W$ N* p4 U# @5 {7 v  B0 |
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It has been reported that a precise level of Oct4 is required to maintain the undifferentiated state of mESCs. The threshold for inducing differentiation is apparently set at 50% above or below the normal diploid expression level in undifferentiated mESCs . We observed that H1T cells express 50% more Oct4 and hold a self-renewal advantage without loss of pluripotency (Table 1; Fig. 1). This observation suggests that the expression level of Oct4 in H1T might function to maintain hESCs properties rather than to induce differentiation. Mouse ESC do not spontaneously differentiate when maintained on mouse embryonic fibroblasts (MEFs) and with LIF. H1 hESC on the other hand do spontaneously differentiate at an approximate rate of 10% in standard conditions (on MEFs and with bFGF). Therefore, this suggests that Oct4 levels in human ESCs would be characteristic of the undifferentiated state at a lower amount. We believe the Oct4 expression in H1T cells favors this undifferentiated condition. Thus, we suggest defining the expression level of Oct4 in H1T cells as 100%. Therefore, the expression level of Oct4 in H1 hESCs maintained on MEF feeder layer is 67%, which is sufficient to maintain the undifferentiated state of hESCs. This also suggests that further modification of the culture system to moderately increase the expression of Oct4 should be considered.

Nodal has been shown to play an important role in early vertebrate development .
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Nodal is able to support prolonged feeder-free culture (more than 10 passages) when it is constitutively overexpressed in hESCs  (Table 1; Fig. 5H, 5I) and thus might block the Nodal function.4 L8 P: @/ M# q& a! R  M

Activin A is expressed both in hESCs and in MEF feeder cells (Table 1; Fig. 5J) . They did not try to do feeder-free culture with only Activin A on Matrigel. We demonstrate that Activin A is able to maintain the long-term feeder-free culture of hESCs on Matrigel at 5 ng/ml (Fig. 3). Thus, we demonstrated a 10-fold lower concentration, which was sufficient for Activin A acting alone to support undifferentiated hESC growth. Our data indicate that although Activin A may not play an important role in early vertebrate development in vivo, Activin A, when present in the in vitro culture of hESCs, could be used to culture hESCs without either feeder cells or conditioned medium. The importance of this finding is that hESCs may be cultured free of feeder cell contamination or serum requirements, which is an important step in identifying a defined medium for culture. The system described here should be useful for generating the large number of hESCs necessary for therapeutic and other applications.
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We report here that a complex signal network contributes to maintain the stemness of hESCs. The signal network includes Nodal/Activin, FGF, Wnt, Hh, BMP, and Notch. Our data indicate that Activin A is both necessary and sufficient to maintain the stemness of hESCs. Our data also suggests that FGF, Wnt, and Hh signaling positively contribute to maintain the self-renewal of hESCs, whereas BMP and Notch signaling negatively regulate the self-renewal of hESCs.3 {- e1 C5 q. d6 j: X- `

There are three pathways, Wnt, FGF, and Nodal/Activin, that have been shown to maintain the self-renewal and pluripotency of hESCs. Our data indicate that Activin A is central for three reasons. First, Activin A induces Oct4 and Nanog at lower concentrations than other factors studied. Activation of Wnt signals only maintained 70% of the Oct4 expression and less than 50% Nanog expression in 6 days compared with CM . In conclusion, we believe that the effect of Activin A on hESCs may be equivalent to the effect of LIF on mESCs to maintain the stemness of embryonic stem cells.5 r! W3 V' Z8 x

ES cells are derived from inner cell mass. During early mouse embryonic development, inner cell mass cells undergo differentiation to a pluripotent cell population termed the epiblast. The epiblast is characterized by expression of a pluripotency marker such as Oct4 and increased expression of fibroblast growth factor 5 . It is interesting that the regulation cascade is conserved between species. In hESCs, our observations indicate that Activin A is able to induce the expression of Oct4, Wnt3, and FGF8 (Fig. 5A, 5C, 5F).
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There is little information about the functions of Notch and Hh in hESCs so far. Our data suggest that Notch might function to induce the differentiation of hESCs and Hh might function to maintain the undifferentiated state of hESCs. The investigations of the function of Notch and Hh in hESCs in the future might provide more insight into the mechanism underlying hESC self-renewal and pluripotency.

DISCLOSURES( c) j+ N. s0 ^8 L4 E1 O9 z

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS

We thank Dr. W. Yu, H. Xu, and L. Suo from the SKCCC Microarray Core Facility for microarray analysis; A. Hawkins and L. Morsberger from Cytogenetics Core Facility for G-banding and SKY analysis; M. Collector and Dr. D. Wang for helpful suggestions; W. Schuler for providing NOD/SCID mice; and the Developmental Hybridoma Bank for providing SSEA3 and SSEA-4 antibodies. We are indebted to Drs. S. Baylin and L. Cheng for helpful review of the manuscript. This work was supported by National Heart, Lung, and Blood Institute, NIH, grants R01-HL54330 and R01-073781.* Z) P$ H! J( O9 M6 K" E: J
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