Defining the Role of Wnt/?-Catenin Signaling in the Survival, Proliferation, and - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

a The Institute for Cell Engineering,! }" U! N# V) O6 ?3 M
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b Departments of Gynecology & Obstetrics and

c Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Key Words. Human embryonic stem cells ? Embryonic stem cell biology ? Self-renewal ? Stem cell differentiation ? Cellular proliferation ? Wnt signaling ? ?-Catenin signaling ? Colony formation assays

Correspondence: Linzhao Cheng, Ph.D., Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Broadway Research Building, Room 747, 733 N. Broadway, Baltimore, MD 21205, USA. Telephone: 410-614-6958; Fax: 443-287-5611; e-mail: lcheng@welch.jhu.edu
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ABSTRACT
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Pluripotent human embryonic stem cell (hESC) lines offer unprecedented opportunities for investigating human cell biology and for developing novel cell-based therapies . All the currently available hESC lines were derived and propagated by coculture with primary mouse embryonic fibroblasts (pMEFs). It was recently found that direct cell-cell contact with pMEFs is not necessary . If an extracellular matrix mixture such as Matrigel is provided, undifferentiated hESCs can be propagated using the conditioned medium (CM) from pMEFs in either serum-containing or serum-free media. Under the latter condition, adding basic fibroblast growth factor (bFGF) and insulin is necessary . However, bFGF is insufficient to prevent hESC differentiation, and the presence of soluble factors made by pMEFs is also required. In contrast to many mouse embryonic stem cell (mESC) lines, adding leukemia inhibitory factor (LIF) and the activation of the gp130/JAK/STAT3 signaling pathway fail to block spontaneous differentiation of hESCs . The nature of soluble factors within pMEF-CM remains undefined, which presents a challenge for improving culture conditions for the propagation (self-renewing proliferation) of hESCs.

Recently, we and others have found that selected human cells, including adult human marrow stromal cells (hMSCs), can substitute pMEFs in supporting the growth of undifferentiated hESCs . After many passages, the expanded hESCs on these supportive human feeder cells continue to form compact colonies of cells expressing markers such as alkaline phosphatase (AP), which is associated with undifferentiated hESCs and mESCs. These studies also found, however, that many human cell types were not supportive: AP-positive (AP ) compact colonies of undifferentiated hESCs disappeared after two passages. The pace of downregulation in expression of AP and other undifferentiated markers in differentiating hESCs is much slower than mESCs , probably due to the fact that hESCs proliferate and differentiate much slower . Therefore, a longer timeframe is required in assays to distinguish undifferentiated or differentiating hESCs. For example, we analyzed hESCs cultured on hMSCs after each passage, which amplified more than 100-fold during the 30-day continuous cultures in five passages . During 2 months in continuous culture, the expanded hESCs expressed a high level of AP and stage-specific embryonic antigen (SSEA)-4, and maintained developmental pluripotency . In the current study, we compared supportive with nonsupportive feeder cells to delineate molecular differences between the two types of feeder cells. We found that all feeder cells, supportive or nonsupportive, produce soluble factors stimulating hESC survival/proliferation. However, cytokines (approximately  30 kDa) secreted from the supportive feeder cells are necessary and sufficient to block differentiation of hESCs when cultured on nonsupportive feeders. Thus, our comparative studies using the two types of feeder cells revealed that an antidifferentiation soluble factor preferentially made by supportive feeder cells is essential to maintain and expand undifferentiated hESCs. The paradigm that an anti-differentiation factor is required to block hESC spontaneous differentiation and to achieve self-renewing proliferation appears similar to that in mESCs, but the antidifferentiation soluble factor for hESCs is not LIF . Because members of the pleiotrophic Wnt cytokine family (~40 kDa) play important roles in many developmental events and affect stem cell fates in several systems , we examined directly whether Wnt is an antidifferentiation factor for keeping hESCs undifferentiated and pluripotent. The results of our extended cell culture (lasting up to 26 days) and functional analyses, however, are contradictory to a recent report concluding that the extracellular Wnt3a signal is sufficient to maintain undifferentiated and pluripotent hESCs, based on the 5-day cell-culture study . To investigate and understand this discrepancy, we decided to further examine the effects of adding Wnt antagonists as well as the same Wnt3a recombinant protein on H1 hESC line as previously described . In addition, we analyzed the Wnt intracellular signaling in hESCs, including the activation of the canonical Wnt signaling pathway mediated by ?-catenin. Based on cell culture and molecular analyses, we propose a new model for the role of Wnt/?-catenin signaling in hESCs.

MATERIALS AND METHODS( N9 ~3 \1 |0 I, d+ G

Establishing a Panel of Feeder Cells with a Different Ability to Support hESCs
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To identify factors made by supportive feeder cells like hMSCs, we compared the ability of fibroblastic hMSCs with other post-natal human fibroblasts such as ccd-1087sk (1087sk, from breast skin) and Hs27 (newborn foreskin) fibroblasts. The latter two displayed morphology and phenotypes similar to hMSCs but lacked the hMSCs’ adiopogeneic, osteogeneic, or chondrogeneic potential . The H1 hESCs were seeded on various irradiated feeders as previously described for the coculture with hMSCs . Because hESCs proliferate and differentiate much slower than mESCs, we examined the fate of hESCs cultured on different feeder cells for several passages by measuring their ability to generate (more) undifferentiated colonies (self-renewing proliferation) in each subsequent passage.

We observed that 1087sk cells as well as hMSCs can support the growth of undifferentiated hESCs as determined by the formation of AP  compact colonies for three consecutive passages (Figs. 1A, 1B). Unlike hMSCs and 1087sk fibroblasts, Hs27 cells could not support the formation of AP  compact colonies (Fig. 1C), an assay used throughout this study to measure the hESC self-renewal after each passage. With the same assay, we found that newborn foreskin BJ and fetal lung WI-38 fibroblasts failed to support hESC growth after two passages (Fig. 1D), consistent with a recent report .. X9 c' k- U( K, N; u
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Figure 1. Identification of a panel of supportive and nonsupportive human feeder cells. (A): Undifferentiated H1 hESCs formed compact colonies after the third passage of culture on 1087sk breast skin fibro-blasts. (B): The resulting H1 hESC colonies stained positive for AP  as those cultured on marrow stromal cells or primary mouse embryonic fibroblasts (all feeder cells were AP–). Scale bars = 100 μm. (C): Cumulative numbers (No) of AP  compact (undifferentiated) colonies over three passages on three types of human feeder cells. The mean and SD (n = 3) are plotted in log scale. (D): The parental 1087sk and the immortalized (HAFi) human cells support growth of undifferentiated hESCs after two passages, whereas Hs27, WI-38, and BJ fibro-blasts do not. Abbreviations: AP, alkaline phosphatase; HAFi, Human Adult Fibroblast, immortalized; hESC, human embryonic stem cell.

To obtain a sustainable source of the human feeder cells, we attempted to immortalize both hMSCs and 1087sk human fibro-blasts by introducing the gene encoding hTERT, the catalytic subunit of the human telomerase. The transduced MSCs acquired a much faster growth rate, became irradiation-sensitive, and were therefore discontinued. The transduced ccd-1087sk cells had the same growth rate even after 42 passages (weekly), whereas the parental cells senesced after 32 passages . We designated the resulting feeder cells as HAFi. HAFi cells can also support the growth of hESCs at least as well as the parental 1087sk cells and pMEFs. The hESCs grown on the HAFi feeders had a characteristic morphology (Figs. 1A, 1B), expressed undifferentiated markers such as SSEA-4, and formed teratomas . Thus, we established a panel of supportive (MSCs, 1087sk, and HAFi) and non- (or poorly) supportive (Hs27, BJ, and WI-38) feeder cells for a unique comparative study.1 h5 ~- @6 E- Q, e, ^5 r

The Supportive Feeder Cells Preferentially Produce a Soluble Antidifferentiation Factor

To determine whether the lack of AP  hESC colonies cultured on nonsupportive cells is due to cell death or differentiation (hESCs are viable but unable to form AP  compact colonies), we used hESCs that were stably transduced with a lentiviral vector expressing the GFP reporter gene. GFP  compact colonies of hESCs were observed 4–7 days after plating onto supportive feeder cells (HAFi or pMEFs) and stained positive for AP (not shown). When Hs27 (nonsupportive) cells were used, GFP  cells were present (Fig. 2Aa), but fewer AP  compact colonies were formed and GFP  cells appeared to be differentiated (Fig. 2Ab).; Q- t8 X& b5 t- }# {

Figure 2. The antidifferentiation activity is present in the CM from supportive feeder cells. (A): Fate of H1 hESCs (GFP ) cultured on nonsupportive Hs27 feeder cells in the presence of basal ESC medium (a, b), pMEF-CM (c, d), or HAFi-CM (e, f) at day 5. GFP  hESCs were traced by GFP fluorescence (upper panels) followed by AP staining (low panels). The presence of GFP  hESCs and loss of AP  compact colonies on nonsupportive feeder cells (a, b) indicate that hESCs survived but differentiated. pMEF-CM (c, d) and HAFi-CM (e, f) restored the AP  compact colony formation (quantified in C). Scale bar = 100 μm. (B): MSC-CM and 1087sk-CM can also restore the ability to support undifferentiated H1 hESCs cultured on Hs27 non-supportive feeder cells. The numbers (No) of AP  compact colonies were enumerated after staining at day 5 (n = 3). (C): Similar activities of pMEF-CM and HAFi-CM on H1 hESCs cultured on Hs27 feeder cells. Fractionation experiments show that activity is retained in the fraction of approximately  30 kDa. Abbreviations: AP, alkaline phosphatase; CM, conditioned medium; GFP, green fluorescent protein; HAFi-CM, Human Adult Fibroblast, immortalized–conditioned medium; hESC, human embryonic stem cell; MSC, marrow stromal cell; MSC-CM, marrow stromal cell–conditioned medium; pMEF-CM, primary mouse embryonic fibroblast–conditioned medium.
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To determine if the antidifferentiation activity of supportive feeder cells is cell-associated or secreted, we tested the effect of CM from supportive MSCs or 1087sk feeder cells on the growth of hESCs cultured on Hs27 feeder cells unable to support hESC growth for two or more passages (Figs. 2Ab, 2B). This experiment may also help to distinguish two possibilities: nonsupportive Hs27 cells are either lacking an antidifferentiation factor (the result should be positive) or producing a dominant differentiation-inducing factor (the result should be negative). We observed that the MSC-CM or 1087sk-CM was sufficient to convert Hs27 feeder cells to support hESCs (Fig. 2B), suggesting that the soluble factor(s) responsible for maintaining undifferentiated hESCs (or antidifferentiation) was present in MSC-CM and 1087sk-CM. Similar results were observed with HAFi-CM and pMEF-CM (Figs. 2Ac–2Af, 2C). The reciprocal experiment gave similar results when the Hs27-CM was applied to supportive feeder cells (not shown). These data suggest that supportive feeder cells produce an extra soluble factor that blocks differentiation of hESCs, in addition to survival/proliferation factors produced by both types of feeders. Initial fractionation of CM indicated that the supportive activity in both HAFi-CM and pMEF-CM resides in the  30-kDa fraction (Fig. 2C)./ B" z% [# a: E, y

Wnt is Not the Antidifferentiation Factor Secreted by the Supportive Feeder Cells
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Comparative gene microarray and proteomic analyses are ongoing to systemically identify genes that are differentially expressed by supportive (vs. nonsupportive) feeder cells. Because the activity in the supportive CM was retained in the fraction of  30 kDa, we assessed the possibility that the pleiotropic secreted protein Wnt (~40 kDa) is the antidifferentiation factor secreted from the supportive feeder cells. The expression of genes encoding several forms of Wnt and its soluble antagonists was detected in both human supportive and nonsupportive feeder cell types (supplemental online Table 1) and in undifferentiated and differentiated hESCs (supplemental online Table 2). To directly test whether Wnt signaling is critical to self-renewing proliferation of hESCs, we used two soluble Wnt antagonists, secreted Frizzled-Related Protein 2 (sFRP2) and Dickoppf-1 (Dkk-1), which block the binding of Wnt to its receptors, Frizzled and coreceptors Low-Density Lipoprotein Receptor Related Protein (LRP)-5 or LRP-6, respectively. Both antagonists were used previously to block diverse functions of the Wnt signaling in vivo or in vitro . hESCs were cultured on HAFi cells in the presence of sFRP2 or Dkk-1 for up to 26 days. The presence of either Wnt antagonist slightly reduced but did not abolish the ability of hESCs to form AP  compact colonies in each subsequent passage (Fig. 3A). The treated hESCs retained the expression of undifferentiated markers such as SSEA-4 after the 26-day treatment (Fig. 3B). Similar results were observed when pMEFs were used as feeder cells (not shown). In addition, adding the purified Wnt3a protein (up to 100 ng/ml) to hESC cultures on nonsupportive Hs27 cells could not substitute the antidifferentiation activity present in the pMEF-CM or HAFi-CM in supporting the formation of AP  colonies (not shown).

Figure 3. Blocking Wnt signaling does not abolish self-renewal of undifferentiated hESCs on supportive feeder cells. GFP  H1 hESCs were cultured on HAFi cells in the presence or absence of Wnt antagonists sFRP2 or Dkk-1 (250 ng/ml). Continuous presence of Wnt antagonists did not abolish the AP  colony-forming ability of hESCs as analyzed at days 5, 8, and 16 (the end of the third passage). (A): The cumulative numbers (No) of AP  compact colonies after each passage are plotted as mean and SD (n = 3). (B): Flow cytometric analysis of H1 hESCs in coculture (identified as GFP  cells) for the expression of SSEA-4 (a marker of undifferentiated hESCs) at day 26, after continuous treatment by the Wnt antagonists. Abbreviations: AP, alkaline phosphatase; GFP, green fluorescent protein; HAFi, Human Adult Fibroblast, immortalized; hESC, human embryonic stem cell; PE, phycoerythrin.
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To block Wnt activities more effectively, we performed the blocking experiment with hESCs cultured on a feeder-free system using Matrigel supplemented with pMEF-CM . Several days after seeding on Matrigel, flattened colonies were observed together with few differentiating cells outside colonies (Fig. 4Aa). The cells harvested from the Matrigel/pMEF-CM culture, however, can form compact AP  colonies again after being seeded on pMEFs (Fig. 4Ab). Therefore, we decided to measure the level of undifferentiated hESCs present in the Matrigel/pMEF-CM culture based on the numbers of compact AP CFUs formed in the subsequent pMEF culture. The latter step allows us to count AP  colonies more accurately and, more importantly, to measure the level of colony-forming, undifferentiated hESCs present in the feeder-free system. The feeder-free system allows us to measure precisely the total numbers of recovered hESCs and their correlation to AP CFUs. When hESCs were cultured on Matrigel (with pMEF-CM) in the presence of sFRP2 or Dkk-1 for 4 days, the total numbers of cells recovered were reduced by 86% and 80%, respectively, as compared with the control pMEF-CM alone (Fig. 4Ba). However, the numbers of AP CFUs were reduced only by 35% and 32%, respectively. Thus, the RF of AP CFUs, a quality index of undifferentiated hESCs, actually increased 4.48-fold and 3.42-fold after treatment of sFRP2 and Dkk-1, respectively.6 I. K* c; n9 V3 O5 U; h6 J4 Z, G3 `
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Figure 4. Effects of adding Wnt antagonists or Wnt3a on H1 and H9 hESCs cultured in the Matrigel feeder-free system. (A): H1 hESCs proliferated and formed flattened colonies on Matrigel at day 4 with pMEF-CM (a). The dissociated cells can form AP  compact colonies again once plated on pMEFs (b). Adding sFRP2 or Dkk-1 to pMEF-CM did not abolish the ability to form AP CFUs (c, d). (B): Relative numbers of harvested H1 hESCs (left) and resulting AP CFUs (right) after first and second passages under various culture conditions. Four days after seeding with equal numbers of hESCs (a, passage 1), total recovered cells in each group (n = 3) were counted and plotted (left panels). One third of the harvested cells were seeded again under the same condition (for passage 2), and one third were seeded on pMEFs to assay for AP CFUs (right panels). For a simple comparison, both parameters were normalized by the mean value of the pMEF-CM control group (defined as 100%). The RF of AP CFUs of each cell population was calculated and normalized by the pMEF-CM group (defined as 1.00, far right). In the absence of pMEF-CM, Wnt3a (100 ng/ml) was added into the ES MED. After passage 2, too few cells survived at passage 2 for the ES MED or the  Wnt antagonist groups. (C): Similar assays with H9 hESCs. (D, E): Morphology of hESCs with the ESC medium alone (ES MED) or Wnt3a at day 4 (a). Representative morphology of AP CFUs (formed subsequently on pMEFs) is also shown in (b), and a colony from differentiating hESCs after Wnt3a treatment is shown in (c). Scale bar = 100 μm. Abbreviations: AP, alkaline phosphatase; AP CFU, alkaline phosphatase–positive colony-forming unit; ES MED, embryonic stem cell medium; ESC, embryonic stem cell; GFP, green fluorescent protein; HAFi-CM, Human Adult Fibroblast, immortalized–conditioned medium; hESC, human embryonic stem cell; pMEF, primary mouse embryonic fibroblast; pMEF-CM, primary mouse embryonic fibroblast–conditioned medium; RF, relative frequency.
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At day 4, equal fractions of the harvested hESCs from the first passage were treated under the same condition after a 1:3 split. After an additional 4 days, sFRP2 reduced cell numbers by 58% compared with the pMEF-CM control, whereas Dkk-1 did not significantly reduce cell numbers (Fig. 4Bb). However, the RF of AP CFUs in the presence of sFRP2 or Dkk-1 was similar to the pMEF-CM control. Similar results were observed at day 13 (not shown). Representative undifferentiated hESC colonies (AP CFUs) grown with or without Wnt antagonists for 13 days are shown in Figure 4A (c, d). Thus, blocking the Wnt signaling significantly reduced the level of hESC survival/proliferation under the feeder-free condition, but the majority of AP CFUs remained. The RF of AP CFUs actually increased after sFRP2 or Dkk-1 treatment, indicating undifferentiated hESCs were likely enriched (Fig. 4B).
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Purified Wnt3a Proteins Are Unable to Substitute for Soluble Factors in pMEF-CM

We next added recombinant Wnt3a proteins to the basal hESC medium to examine whether it could replace pMEF-CM in the freeder-free Matrigel culture. In the absence of pMEF-CM, only small clusters of hESCs remained after 4–5 days with the hESC medium alone (Fig. 4D). The addition of Wnt3a (up to 100 ng/ml) into the basal medium (containing bFGF and insulin) provided a cell survival/proliferation advantage in a dose-dependent manner (not shown). After Wnt3a treatment of 4–5 days, surviving hESCs formed fewer, but more compact, colonies as compared with the culture with pMEF-CM (Fig. 4E), similar to the previous report . We did not further examine the role of Wnt on the morphological changes, although it has been reported that Wnt also affects cell adhesion and planar polarity . Instead, we harvested the surviving hESCs in the presence or absence of Wnt3a after the first passage and assayed for AP CFUs. As compared with the pMEF-CM control (taken as 100%), 52% and 20% of hESCs could be recovered when Wnt3a was present or absent in the ESC medium (Fig. 4B, left panel, red bars). The Wnt3a stimulatory effect was largely blocked by sFRP2 or Dkk-1 (blue and purple bars), and the cell numbers reduced to the minimal levels both in the presence or absence of exogenous Wnt3a. Collectively, the data indicate that sFRP2 and Dkk-1 blocked the stimulatory effect of survival/proliferation by autocrine Wnt signaling in hESCs (more obvious when the basal medium alone was used) as well as paracrine Wnt signaling (when pMEF-CM or exogenous Wnt was used). Despite the significant increase in total cell numbers, adding Wnt3a neither increased AP CFUs proportionally nor substituted for pMEF-CM in maintaining AP CFUs. When pMEF-CM was absent, the RF of AP CFUs was actually decreased to 0.29 when Wnt3a was present (Fig. 4B).% j8 C* Z& A. t2 a' U8 D& D

We further examined the Wnt3a effects beyond the first passage (4–5 days), as we did with pMEF-CM (Fig. 4Bb). Too few cells survived in the group without Wnt3a after an additional 4 days, preventing further analysis. In the presence of Wnt3a, the numbers of recovered cells and AP CFUs were reduced further to 31% (accumulatively 16%) and 2.6% (accumulatively 0.4%), respectively, as compared with the pMEF-CM control group (100%). The RF of AP CFUs in the Wnt-treated cells reduced to 0.08 at passage 2 (Fig. 4Bb), 12.5-fold lower than the pMEF-CM control.
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In addition to the H1 hESCs we used extensively, we also examined the Wnt3a effects on a different hESC line, H9 (Fig. 4C). Although the growth properties of H9 are not identical to H1, we also observed that Wnt3a modestly enhanced H9 hESC survival/proliferation and colony formation at day 4. However, at the end of the second passage (day 8),  5% of colonies formed if Wnt3a was used to replace pMEF-CM. Similar to H1, the RF of AP CFUs in the Wnt-treated H9 cells was reduced to 0.17 at passage 2 (Fig. 4Cb), sixfold lower than the pMEF-CM control.. z$ ^0 H1 u: K' z+ }8 V9 k9 M) Z
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To examine molecular changes following the Wnt3a treatment, we assayed the expression of marker genes that are preferentially expressed in either undifferentiated or differentiated ESCs. As a control, we also used hESCs after treatment by adding two known differentiation-inducing agents, BMP4 or RA, into pMEF-CM. Consistent with a previous report , hESCs treated with BMP4 quickly differentiated (Fig. 5A). Cells treated with RA also differentiated although at a much slower rate . By day 9, the harvested cells with BMP4 or RA treatment contained few AP CFUs (Fig. 5B). Consistent with a previous report , the level of Oct-4 mRNA in the BMP4-treated cells was reduced by more than fivefold (Fig. 5C). The RA-treated sample, which lost the AP CFU biological activity at day 9, showed marginal reduction in Oct-4 mRNA (Fig. 5C). The observation in the differentiated hESCs induced by RA is consistent with the notion that Oct-4 expression is not sufficient and that other signals in the "stem cell orchestra" are also required to maintain undifferentiated ESCs . In Wnt3a-treated cells (when pMEF-CM was absent), the level of Oct-4 mRNA was reduced by 50% (Fig. 5C). We next examined the expression of genes such as Brachyury, the expression of which is associated with lineage commitment/differentiation (Fig. 5D). The expression of the Brachyury gene is transiently activated at the onset of mesoderm formation and also possibly the endoderm lineage in the mouse system, and is regulated by Wnt3a . Indeed, the expression of the Brachyury gene was significantly increased in the Wnt3a-treated cells as well as in differentiated cells induced by BMP4 and RA (Fig. 5D). Based on both AP CFU formation assays and RT-PCR analysis, we conclude that Wnt3a accelerates hESC differentiation (and cell survival/proliferation) when an antidifferentiation factor is absent.% b" a  C5 {2 }2 |5 A7 H

Figure 5. Analysis of differentiating hESCs induced by various culture conditions. (A, B): Colony-forming ability of H1 hESCs after treatment with BMP4 (10 ng/ml), RA (10–6M), or DMSO (solvent control for RA) at day 4 and 9, respectively. The mean and SD (n = 3) of relative frequencies of AP CFUs are plotted accordingly after being normalized by the mean value of the pMEF-CM group (defined as 1.00). (C, D): Quantitative reverse transcription–polymerase chain reaction analyses to measure mRNA levels of the Oct-4 gene (C) and the Brachyury gene associated with mesoderm and endoderm differentiation (D). In addition to the BMP4- and RA-treated samples harvested at day 9, the Wnt3a-treated sample (ES MED Wnt) at day 8 (Fig. 4B) was also included. After reverse transcription, cDNA from each sample was amplified for 45 cycles with primers specific for Oct-4 or Brachyury gene and the ?-actin gene used an internal control. The relative level of Oct4 or Brachyury mRNA was first normalized by that of ?-actin in each sample (n = 4). For a simple comparison, the mean value of relative mRNA level in the pMEF-CM control group is defined as 1.00, either for Oct-4 (C) or Brachyury (D). Note that Oct-4 mRNA is expressed at a high level (C) and Brachyury is barely detectable (D) in the pMEF-CM control group. Abbreviations: AP CFU, alkaline phosphatase–positive colony-forming unit; BMP-4, bone-morphogenetic protein 4; DMSO, dimethyl sufoxide; ES MED, embryonic stem cell medium; hESC, human embryonic stem cell; pMEF-CM, primary mouse embryonic fibroblast–conditioned medium; RA, retinoic acid.

Direct Examination of Wnt Downstream Effector Molecules Inside hESCs

mRNAs of multiple genes involved in the Wnt signaling pathway were detected in both undifferentiated and differentiated H1 hESCs in our microarray analyses (supplemental online Table 2), similar to data reported for various hESC lines. These include several forms of Wnt antagonist genes such as sFRP1, sFRP3, Dkk-1, Dkk-2, Dkk-3, and WIF-1 as well as Wnt5a. Genes encoding known downstream molecules of the Wnt canonical signaling pathway such as GSK-3? , ?-catenin, and the family members of TCF/lymphoid enhancer-binding factor (LEF) transcriptional factors are also expressed in both undifferentiated and differentiated hESCs (supplemental online Table 2). In the canonical Wnt signaling pathway, a key event is that tightly regulated ?-catenin enters nucleus and transiently activates TCF, which in turn binds its cognate DNA sequence in the regulatory region of dozens of Wnt/?-catenin target genes. To directly measure the Wnt/?-catenin canonical signaling in hESCs, we used an improved version of the TOP flash (TOP) reporter system , in which the luciferase reporter gene activity is controlled by the ?-catenin/TCF transcriptional activation. After transfection, the luciferase activity in undifferentiated or differentiating hESCs is measured, reflecting the internal status of the Wnt/?-catenin activation.
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We detected a low level of ?-catenin/TCF–mediated activation in undifferentiated H1 and H9 hESCs maintained by pMEF-CM (Fig. 6). Adding Wnt3a into pMEF-CM significantly increased the TOP activity, consistent with previous data that hESCs are responsive to Wnt stimulation. In differentiating hESCs after BMP4 or RA induction, or Wnt3a alone (without pMEF-CM), the TOP activity was also significantly increased (Fig. 6A). The late-passage H1 hESCs (p79–85) showed a higher level of basal TOP activity (Fig. 6B) than the earlier-passage H1 (p41–45; Fig. 6A) or H9 cells (p29–34; Fig. 6C). In addition, both basal (if any) and Wnt3a stimulatory TOP activities in H1 (Fig.6B) and H9 (Fig. 6C) hESCs can be fully blocked by the Dkk-1 inhibitor. Taken together with the AP CFU and RT-PCR analyses (Fig. 5), our data indicate that the ?-catenin/TCF–mediated activation in undifferentiated hESCs is minimal under the standard culture condition using pMEF-CM. However, it is significantly elevated during initial hESC differentiation induced by several different methods. Thus, the Wnt/?-catenin activation is not indicative of the undifferentiated (and pluripotent) state of hESCs.! B8 s9 B' b9 `5 o+ B9 d; C* Q
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Figure 6. The Wnt/?-catenin–mediated transcriptional activation is minimal in H1 and H9 hESCs and significantly elevated after differentiation induction. The transcriptional activation activity of ?-catenin in hESCs is measured after transfection of a luciferase reporter (TOP) containing the TCF biding sites. A control (FOP) containing defective TCF sites was also used in parallel assays. Enriched hESCs were plated on Matrigel and cultured in the optimal medium (pMEF-CM). After transfection, cells were cultured in either pMEF-CM or the plain ESC medium (ES Med) with various factors for 2 days. The mean and SD (n = 2) of the luciferase activity from the TOP or FOP reporter were calculated. For a simple comparison, the mean value of the FOP control in the pMEF-CM control group is defined as 1.00, and relative reporter activity is plotted for each condition. Note that the FOP activity is minimal and similar in all the samples. (A): The TOP and FOP activities in H1 hESCs (p41–45) after 2-day treatment with Wnt3a (100 ng/ml), BMP-4 (10 ng/ml), and RA (10–6 M) as in Figure 5. (B): The TOP and FOP activities in late-passaged H1 hESCs (p79–85) after treatment by Wnt3a and its soluble antagonist Dkk-1 (250 ng/ml). (C): The TOP and FOP activities in H9 hESCs (p29–34). H1 (B) and H9 (C) hESCs were treated similarly as indicated by sample numbers below the bar graphs: #1–3 in the PMEF-CM, and #4–6 in ES Med, #2 and #4 with Wnt3a alone, #3 and #6 with Wnt3a and Dkk-1, as compared with no addition (#1 and #4). Abbreviations: BMP-4, bone-morphogenetic protein 4; ES Med, embryonic stem cell medium; FOP, superFOPflash; hESC, human embryonic stem cell; pMEF-CM, primary mouse embryonic fibroblast–conditioned medium; RA, retinoic acid; TCF, transcriptional factor; TOP, superTOPflash.

Wnt3a Directly Stimulates hESC Proliferation
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In the absence of pMEF-CM, we observed more hESCs when Wnt3a was present (ESC medium Wnt) than when Wnt was absent for 5 days (Fig. 4B). This could be due to either a decrease in apoptosis or an increase in hESC proliferation, or both. To distinguish these two possibilities, we performed Annexin-V staining to detect apoptotic cells and the BrdU incorporation to detect DNA synthesizing/proliferating cells under above culture conditions. The results showed that the Wnt addition did not significantly affect the rate of apoptosis, since percentages of apoptotic cells were similar (Figs. 7A–7D). However, adding Wnt3a increased the percentage of, and level in, cells incorporating BrdU (Fig. 7G), as compared with the basal ESC medium group (Fig. 7F). Therefore, Wnt can directly enhance hESC cycling and proliferation with little effect on preventing apoptosis.0 r8 i  n. F; L& n

Figure 7. Wnt activation stimulates hESC proliferation and has little effect on apoptosis. (A–D): Flow cytometric analysis of apoptotic cells using Annexin-V surface staining and 7 AAD staining (for DNA in permeable dying cells). Shown are dot plots of the FITC-conjugated Annexin-V (Annexin FITC) versus 7 AAD staining for hESCs cultured in pMEF-CM (A), the basal ESC medium (B), and ESC medium Wnt3a (C). The percentages of dying cells in the early stage (Annexin /7AAD nonpermeable) and late stage (Annexin /7AAD permeable) are indicated in the lower and upper right quadrants, respectively. The relative percentages are summarized in (D). (E–H): Wnt3a activation stimulates hESC proliferation. The percentages of hESCs incorporating BrdU (actively cycling) versus DNA content (stained with 7AAD after permeation step) are shown when cultured with pMEF-CM (E), the basal ESC medium (F), or ESC medium Wnt3a (G). (H): Cells cultured with pMEF-CM and treated as in (E), but BrdU pulse was omitted to assess nonspecific staining by the anti-BrdU antibody. Apoptotic cells that are stained falsely positive by the anti-BrdU antibody are indicated by an asterisk. Abbreviations: ESC, embryonic stem cell; FITC, fluorescein isothiocyanate; hESC, human embryonic stem cell; pMEF-CM, primary mouse embryonic fibroblast–conditioned medium.

DISCUSSION

This work was supported in part by research grants from the National Institutes of Health (HL73781), Leukemia and Lymphoma Society of America (6189-2), and the Johns Hopkins Institute for Cell Engineering. We thank Drs. Qian Xu and Jeremy Nathans (Johns Hopkins University ) for providing advice and reagents related to the Wnt signaling pathways, Dr. Randall Moon (HHMI in University of Washington) for allowing us to use the SuperTOPflash and superFOPflash reporter system, and Stephanie Kueng (JHU) for technical support. We also thank Drs. John Gearhart, Hongjun Song, and Leslie Lock (JHU) for critical reading of the manuscript. G.D. and Z.Y. contributed equally to this work.

DISCLOSURES
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The authors indicate no potential conflicts of interest.

REFERENCES7 D7 A) L" G0 ?( w6 M/ P

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147." u3 `1 ?# z" X+ Q' J

Reubinoff BE, Pera MF, Fong C et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.! Q0 _$ E  Z) J6 I' S
+ @. W: w2 U, h2 c) J+ t
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.

Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–278.
4 o* M! `, }- G( ^" J
Daheron L, Opitz SL, Zaehres H et al. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. STEM CELLS 2004;22:770–778.
/ _1 o7 ?! g" r. {
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
4 u0 l8 H" k& A. @8 N
Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.7 e# T) J5 ^$ m  N8 s) z/ I
, m- N3 t7 G3 S
Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.
% x1 b6 O6 M. {6 A9 @  P5 r
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
$ Y$ {: L9 }* L# D6 C
Richards M, Tan S, Fong CY et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. STEM CELLS 2003;21:546–556.

Draper JS, Pigott C, Thomson JA et al. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 2002;200:249–258.
8 @. I' P2 o& f9 i
Moon RT, Kohn AD, De Ferrari GV et al. WNT and beta-catenin signaling: diseases and therapies. Nat Rev Genet 2004;5:691–701.
7 f# K. X. w4 G) ]- i
Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science 2004;303:1483–1487.

Kleber M, Sommer L. Wnt signaling and the regulation of stem cell function. Curr Opin Cell Biol 2004;16:681–687.# t9 Q9 r) C8 K7 L9 g. b0 G
7 ~2 @+ C$ _. p; P
Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.' p7 j8 H! D# S) c: }8 p5 R# L

Dravid G, Hammond H, Cheng L. Culture of human embryonic stem cells on human and mouse feeder cells. In: Turksen K, ed. Human Embryonic Stem Cells. Totowa, NJ: Humana Press, (in press).
9 i( [- V  c; X
Cui Y, Golob J, Kelleher E et al. Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood 2002;99:399–408.

Aubert J, Dunstan H, Chambers I et al. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 2002;20:1240–1245.
& C5 i, q7 h5 o# {
Polesskaya A, Seale P, Rudnicki MA. Wnt signaling induces the myogenic specification of resident CD45  adult stem cells during muscle regeneration. Cell 2003;113:841–852.

Gregory CA, Singh H, Perry AS et al. The Wnt signaling inhibitor dick-kopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 2003;278:28067–28078.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the  CT method. Methods 2001;25: 402–408.' b, k0 u6 b7 P7 W# Z
* L( }# e  Q( }( ]1 G: X# X
Veeman MT, Slusarski DC, Kaykas A et al. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol 2003b;13:680–685.

Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 2000;284:143–147.

Xu RH, Chen X, Li DS et al. BMP-4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 2002;20:1261–1264.; }" O, b$ ?0 D5 Z" X& M" r
1 R0 R2 N, O0 G6 ?; g7 d
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.2 H0 Q' u4 T! G6 L3 j$ a& e# B
# ]6 o, Y% [/ }& x1 n
Cavaleri F, Scholer HR. Nanog: a new recruit to the embryonic stem cell orchestra. Cell 2003;113:551–552., R7 F. C" N& f4 F0 Y) \* {: q

Yamaguchi TP, Takada S, Yoshikawa Y et al. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 1999;13:3185–3190.* ^$ ?4 B+ a% P& Y& j8 H
" j2 Z! X1 P% a6 t
Arnold SJ, Stappert J, Bauer A et al. Brachyury is a target gene of the Wnt/beta-catenin signaling pathway. Mech Dev 2000;91:249–258.1 ^9 ~! s7 |7 \9 P
1 R6 t3 U. s! Q. f
Kubo A, Shinozaki K, Shannon JM et al. Development of definitive endoderm from embryonic stem cells in culture. Development 2004;131: 1651–1662.

Xu RH, Peck RM, Li DS et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005;2:185–190.

Beattie GM, Lopez AD, Bucay N et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. STEM CELLS 2005;23:489–495.
4 I5 f8 O- K: H" f- [
Meijer L, Skaltsounis AL, Magiatis P et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003;10:1255–1266.
. ?# ?: X3 {8 Q1 j
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.
# Z! y' {- ^4 Y4 ?$ {
Xu C, Rosler E, Jiang J et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. STEM CELLS 2005;23:315–323.! V1 a8 s4 W4 \, \  u7 E
$ |/ v  h& i# h' F
Wang L, Li L, Menendez P et al. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 2005;105:4598–4603.3 a3 v7 h( Y( V' S  d6 s' w
: d1 e0 K7 {3 n3 ]
Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.! A$ i1 m3 `' a- f8 L

Kielman MF, Rindapaa M, Gaspar C et al. Apc modulates embryonic stem-cell differentiation my modulating the dosage of beta-catenin signaling. Nat Genet 2003;32:594–605.2 U0 k1 z% b: C, F9 d% U
( @" r  w) H, o$ |
Otero JJ, Fu W, Kan L et al. Beta-catenin signaling is required for neural differentiation of embryonic stem cells. Development 2004;131:3545–3557.0 [; E' o9 q, L  F" l: ]
5 f5 ~2 l9 T+ b0 ]
Veeman MT, Axelrod JD, Moon RT. A second canon: functions and mechanisms of ?-catenin-independent Wnt signaling. Dev Cell 2003b;5:367–377.: g) U8 }' A( \; s* S0 \+ D. F2 W

Huelsken J, Vogel R, Brinkmann V et al. Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol 2000;148:567–578.
! o! b1 g( y2 Q, `. ^( u. A0 _
Cobas M, Wilson A, Ernst B et al. Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. J Exp Med 2004;199:221–229.4 n$ ?6 W1 u. @) [

Lee HY, Kleber M, Hari L et al. Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 2004;303: 1020–1023.

Reya T, Duncan AW, Ailles L et al. A role for Wnt signaling in self-renewal of haematopoietic stem cells. Nature 2003;423:409–414.
$ P/ q( B$ A/ `% X
Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448–452.

Alonso L, Fuchs E. Stem cells in the skin: waste not, Wnt not. Genes Dev 2003;17:1189–1200.7 f! j) E9 I! @4 `1 P* ?6 J) J
3 z8 o/ L- Z1 l$ B+ ^/ A# L
Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359–363.
- v; b0 p2 ~8 i+ k% [( _6 R2 `; l
He XC, Zhang J, Tong WG et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet 2004;36:1117–1121.(Gautam Dravida,b, Zhaohui)

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