EMBRYONIC STEM CELLS Notch Signaling Is Inactive but Inducible in Human Embryonic Stem Cells SCOTT A. NOGGLE,a,c DEBORAH WEILER,b BRIAN G. CONDIEa c
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA; bBresaGen Inc., Athens, Georgia, USA; aDepartment of Genetics, University of Georgia, Athens, Georgia, USA
Key Words. Differentiation • ␥-Secretase • Human embryonic stem cell • Human development • NOTCH intracellular domain HES
ABSTRACT The NOTCH signaling pathway performs a wide range of critical functions in a number of different cell types during development and differentiation. The role of NOTCH signals in human embryonic stem cells (hESCs) has not been tested. We measured the activity of canonical NOTCH signaling in undifferentiated embryonic stem (ES) cells and tested the requirement for NOTCH activity in hESC self-renewal or differentiation by growing hESCs in the presence of ␥-secretase
inhibitors. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of the differentiating cell types that accumulate in human ES cell cultures. Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors. STEM CELLS 2006;24:1646 –1653
INTRODUCTION
naling pathways involved in generating and maintaining the differentiated cell types is a key to controlling differentiation to form specific cell types. The NOTCH signaling pathway directs a multitude of functions during development [10 –12]. Because previous work has shown that the mRNAs encoding NOTCH1–3 and DLL1 were present in human ES cells [13], we tested the role of NOTCH signaling in hESCs and differentiating cells derived from hESCs. Our results indicate that NOTCH can be activated in hESCs by transiently exposing the cells to cation chelators (trypsin-ethylenediaminetetraacetic acid [EDTA]). This result suggests that NOTCH signaling is activated in human ES cells by cell passaging conditions that include cation chelation. Although NOTCH signaling is inducible in hESCs, we find that it is normally inactive in undifferentiated cells. However, inhibition of NOTCH signaling with a ␥-secretase inhibitor markedly reduces the number of differentiating cells in human ES cultures, suggesting that the NOTCH pathway is only required for the maintenance cell types. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of many of the differentiating cell types that accumulate in human ES cell cultures. We also found that NOTCH activity appears to dramatically increase in differentiated cells derived from the hESCs. The level of nuclear NOTCH intracellular domain (NICD) protein is greatly upregulated in the differentiated cells.
A key step in controlling human embryonic stem cell (hESC) differentiation is defining the pathways that function in undifferentiated cells and the ways in which culture conditions and extrinsic factors can activate or repress them. Current evidence supports fundamental roles for fibroblast growth factor (FGF) and WNT signaling in the maintenance of undifferentiated hESCs [1–3]. In addition, factors that can activate SMAD2/3 activity, such as members of the transforming growth factor-/nodal/activin family appear to act downstream of FGF or WNT signals to maintain hESC pluripotency [4 – 6]. Defining these key players in maintaining the undifferentiated state has led to the development of new methods to culture hESCs and has uncovered fundamental differences in the biology of human and mouse ES cells [7]. Another aspect of understanding hESC growth and differentiation is defining the signaling pathways required for the formation of differentiated cell types. As ES cells differentiate to form the various stem cells of the developing embryo, additional pathways supplant or interact with those active in undifferentiated cells. Differences in signaling pathways between undifferentiated ES cells and neural precursors have been exploited to remove undifferentiated cells from ES-derived neural cell cultures, reducing the teratoma-forming ability of the highly heterogeneous mixture of cells in embryoid bodies [8]. In addition, hESC cultures can accumulate a population of differentiated cells over time, particularly in cultures maintained by cell dispersal [9]. Identification of the sig-
Correspondence: Brian G. Condie, Ph.D., Department of Genetics, University of Georgia, Davison Life Sciences Complex, Athens, Georgia 30602, USA. Telephone: 706-542-1431; Fax: 706-583-0691; e-mail:
[email protected] Received July 12, 2005; accepted for publication March 21, 2006; first published online in STEM CELLS EXPRESS April 13, 2006; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0314
STEM CELLS 2006;24:1646 –1653 www.StemCells.com
Noggle, Weiler, Condie Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors.
MATERIALS
AND
METHODS
Cell Culture and ␥-Secretase Inhibition The hESCs used in this study are approved for NIH-funded research. BGN01 (BG01), BGN02 (BG02), and H1 (WA01) were maintained on mouse embryonic fibroblasts (MEFs) and passaged by microdissection. For the ␥-secretase experiments, collagenase type IV plus trypsin-EDTA was used to expand the hESCs for no more than 10 passages before stage-specific embryonic antigen (SSEA) 4 selection. Cells were treated with 1 mg/ml collagenase (Gibco, Grand Island, NY, http://www. invitrogen.com) in Dulbecco’s modified Eagle’s medium (DMEM), 20% Knockout Serum Replacement (KSR), 1% minimal essential medium nonessential amino acids, 1 mM Lglutamine, penicillin (0.5 U/ml), streptomycin (0.5 U/ml), and 0.1 mM -mercaptoethanol for 3–5 minutes. The collagenase was removed, and 0.05% trypsin-EDTA was added for 45 seconds. Trypsin was neutralized with DMEM containing 10% fetal bovine serum and 10% KSR, and cells were washed in growth medium containing 8 ng/ml basic FGF and plated at 20,000 cells per cm2 on MEFs. Cells were enriched by magnetic cell sorting (MACS) as described previously [14]. Cells were incubated in 5 M N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycine t-butyl ester (DAPT) in 0.5% dimethyl sulfoxide (DMSO), or 0.5% DMSO alone as a control to test the effect of ␥-secretase inhibition. DAPT was purchased from Calbiochem (San Diego, http://www.emdbiosciences.com). There is a great deal known about the specificity and molecular mechanism of various ␥-secretase inhibitors, including DAPT. This is due to their potential utility as drugs for the treatment of Alzheimer’s disease. In the case of DAPT, it has been shown that it inhibits ␥-secretase activity by binding to presenilin 1 [15, 16]. DAPT either binds to the active site directly or binds nearby [15–17]. It is a potent NOTCH inhibitor with an IC50 of 10 nM in a cell-free microsome-based assay using a NOTCH reporter as substrate [17]. In mammalian cells, inhibition of NOTCH cleavage is achieved at 10 – 40 nM [17]. In terms of specificity, DAPT treatment has been shown to produce phenocopies of Drosophila and zebrafish Notch pathway mutations [17–19]. In addition, it has been used as a specific inhibitor of ␥-secretase in numerous studies [20 –23]. Although presenilin is also involved in modulating the WNT signaling pathway, this function appears to be independent of its ␥-secretase activity [24, 25].
Immunofluoresence To detect cell surface antigens, cells were fixed in 4% paraformaldehyde (PFA), 4% sucrose in phosphate-buffered saline (PBS), pH 7.4, and blocked in 3% goat serum, 1% polyvinylpyrollidone in PBS. SSEA4 (1:100; Chemicon, Temecula, CA, http://www. chemicon.com) and NOTCH1 (H-134, 1:10; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt. com) antibodies were added and incubated 2 hours to overnight. Alexa488 antimouse antibodies or Alexa594 anti-rabbit secondary antibodies (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) were incubated for 30 minutes to 1 hour at room temperature. Cell nuclei were stained with DAPI. All washes were in PBS. www.StemCells.com
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Images were captured with a Nikon TE-2000E microscope (Nikon) with a Retiga 1300 cooled CCD camera and Qcapture software (Qimaging, Burnaby, BC, Canada, http://www. qimaging.com). To detect the NOTCH1 NICD in differentiated cells, BG01 hESCs were passaged by microdissection to MEFs as above and grown for 5 days under nondifferentiating conditions. The samples were processed for immunocytochemistry detection of NICD using the cleaved NOTCH1 (Val1744) antibody (Cell Signaling Technology, Beverly, MA, http://www.cellsignal. com) according to the manufacturer’s protocol. The cells were fixed in ice-cold methanol, blocked, and exposed to a 1:500 dilution of primary antibody overnight at 4°C. The cells were washed extensively, and primary antibody was detected with a 1:500 dilution of goat anti-rabbit horseradish peroxidase (HRP)conjugated secondary antibody (Molecular Probes, Inc.) for 2 hours at room temperature. The HRP reaction was developed with the DAB kit (Vector Labs, Inc., Burlingame, CA, http://www. vectorlabs.com) according to the manufacturer’s instructions. Reactions omitting the primary antibody were used as controls.
RNA Analysis Total RNA was prepared with Trizol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and treated with DNAse I. Reverse transcription was performed with oligo(dT) and SuperScript II (Invitrogen). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to measure HES1 transcripts. The cDNA was generated from triplicate samples from three independent experiments. TaqMan PCR Master Mix (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) was used in 25-l reactions with primers at a final concentration of 500 nM and the specific probe at a final concentration of 200 nM. Parallel amplifications of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used for normalization. GAPDH primers were purchased from Applied Biosystems. The primers/probe for HES1 were as follows: Forward, 5⬘-CTACCCCAGCCAGTGTCAAC-3⬘. Reverse, 3⬘-TCAGCTGGCTCAGACTTTCA-3⬘. Probe, 5⬘-CGACACCGGATAAACCAAAGACAGC-3⬘. Real-time RT-PCRs were run on an ABI7700 Sequence Detection System (Applied Biosystems, Inc.). Data were analyzed with REST-XL (version 2) software to determine significant differences in relative expression.
Flow Cytometry Cells were washed once in PBS and fixed in 2% PFA for 20 minutes at room temperature. The cells were blocked in 3% goat serum in PBS. SSEA4 (1:100; Chemicon) and NOTCH1 (H134, 1:10; Santa Cruz Biotechnology) antibodies were added in blocking solution and incubated 2 hours to overnight at 4°C. Alexa488-conjugated anti-mouse antibodies or PE-conjugated anti-rabbit antibodies (Molecular Probes) were used for detection. In some cases, cells were postfixed in 0.5% PFA and washed in PBS, 0.05% azide. Cells were analyzed on a Cytomation CyAn flow cytometer equipped with UV, 488-nm, and 633-nm laser excitation sources. Single stains, isotype controls for directly conjugated primary antibodies, and controls in which primary antibody was omitted in indirect staining protocols were all used for analog
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Figure 1. NOTCH expression in human embryonic stem cells (hESCs). (A, B): Detection of NOTCH1 protein (A) and SSEA4 (B) on a passage 12 BGN1 hESC colony. (C): Western blot analysis of NOTCH pathway proteins. Cleaved NOTCH1 and NOTCH2, as well as processed nicastrin and presenilin proteins, were detected. The Notch intracellular domain was detected since Notch cleavage was activated by calcium chelation due to the use of trypsinethylenediaminetetraacetic acid (EDTA) in harvesting the cells. (D): Reverse transcription-polymerase chain reaction analysis of NOTCH mRNAs. The control lanes are ⫺T, no template and ⫺RT, no reverse transcriptase. Scale bars (A, B) ⫽ 10 m. Abbreviations: CTF, C-terminal fragment; holo, holoprotein; Immat., immature; kD, kDa; Mat., mature; MEF, mouse embryonic fibroblast; ⫹RT, reverse transcriptase; ⫺RT, control, no reverse transcriptase; ⫺T, control, no template.
and postacquisition compensation. In most cases, 10,000 to 50,000 events were collected. Analysis was performed with Summit analysis or FlowJo software.
Western Blotting For blotting, equal amounts of protein were resolved on precast gels (Gradipore), transferred to nylon membranes, and blocked in Tris-buffered saline, 0.1% Tween 20, and 5% milk. Antibodies were as follows: 1:10 bTAN20 (NOTCH1, DSHB) and C651.6bHN (NOTCH2, DSHB); 1:100 anti-Oct4 (C-10; Santa Cruz Biotechnology); 1:1,000 anti-presenilin and nicastrin (Chemicon), cleaved NOTCH1 Val1744 antibody (Cell Signaling), and 0.5 g/ml anti-histone deacetylase (Zymed). Membranes were incubated with primary antibodies overnight and washed in Tris-buffered saline, 0.1% Tween 20. Secondary antibodies were diluted in blocking buffer and incubated with the membranes for 45 minutes at room temperature. Washes were in Tris-buffered saline. Proteins were detected by enhanced chemiluminescence (GE Healthcare Life Sciences, Piscataway, NJ, http://www.amersham.com).
with the control plasmid pRL-CMV (Promega). In some cases, a plasmid expressing NICD (GmN1-ICV-wt, from Dr. R. Kopan, Molecular Biology and Pharmacology, Washington University, St. Louis) was included as a positive control. The hESCs were grown in feeder-free conditions [27], plated on Matrigelcoated 12-well plates, and grown for 2 days prior to transfection. For each transfection, 500 ng of reporter in a total of 1.6 g of DNA was mixed with 4 l of Lipofectamine 2000 (Invitrogen) in 200 l of OptiMEM. This was added to the hESC medium on the cells and incubated overnight. The medium was changed the next morning with MEF-conditioned medium replaced with nonconditioned medium to induce differentiation in some samples. Cells were harvested after 48 or 72 hours for luciferase assay. Transfection efficiency varied with different cell lines. In control transfections, we found that 5%–10% of the cells in culture of H1 hESCs and approximately 15% of the cells in BG01 cultures were transfected.
RESULTS
Luciferase Assays
Key Components Required for NOTCH Signaling Are Present in Undifferentiated hESCs
To detect NOTCH activity, reporter plasmids were transfected into undifferentiated hESCs or hESCs differentiated by withdrawal of conditioned medium. The wild-type (4xwtCBF1Luc) and mutant (4xmtCBF1luc) reporters [26] were cotransfected
We initiated our analysis by confirming that key protein components of the NOTCH pathway are indeed expressed in the hESC lines BGN01 and BGN02. We find that the NOTCH1 receptor is expressed on undifferentiated hESCs (Fig. 1A, 1B).
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Figure 2. Western analysis of NOTCH activation and pathway activity in human embryonic stem cells (hESCs). (A): Notch intracellular domain (NICD) was detected after exposure to trypsin-EDTA (⫹EDTA) but not after collagenase treatment (⫺EDTA). The bTAN20 antibody detects the extracellular domain (120 kDa) and the NICD, whereas the cleaved NOTCH1 antibody only detects NICD. The protein in the middle lane was extracted from a culture plated at a low density on feeders. (B): Inhibition of NOTCH cleavage by a ␥-secretase inhibitor. Human ES cells grown on mouse embryonic fibroblast (MEF) feeders were first incubated for 3 days in a potent ␥-secretase inhibitor, DAPT, or the DMSO carrier and then harvested with trypsin-EDTA. The asterisk indicates a protein detected by the antibody in total protein from MEF feeder cells but not in total protein from hESCs grown without feeders. (C): Real-time reverse transcription-polymerase chain reaction analysis of endogenous HES1 transcripts in undifferentiated hESC cultures. HES1 expression is induced by EDTA, and this induction is blocked by the ␥-secretase inhibitor DAPT. (D): NOTCH activity in undifferentiated and differentiating hESCs. wt4xCBF1-luc or mt4xCBF1-luc was transfected into cells as indicated (⫹ or ⫺). Cells were maintained with (CM⫹) or without (CM⫺) feeder-conditioned medium. Some of the samples were also transfected with an NICD expression vector (NICD⫹). (E): A control transfection demonstrating that NICD can only activate the wildtype CBF1-luc reporter. Abbreviations: CM, conditioned medium; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tbutyl ester; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; HDAC, histone deacetylase; kD, kDa; mt4xCBF1-luc, mutant CBF luciferase reporter; NICD, Notch intracellular domain; wt4xCBF1-luc, wildtype CBF luciferase reporter.
In addition, the NOTCH1 and NOTCH2 proteins are expressed on hESCs, as determined by Western blot analysis (Fig. 1C). In this analysis, we used antibodies directed against the cytoplasmic domain of NOTCH 1 and 2, and in both cases, two bands were detected. The larger, 120-kDa band corresponds to the transmembrane domain of the NOTCH receptor (Notch transmembrane domain), whereas the smaller, 110-kDa band corresponds to the NICD, which is the active intracellular fragment that mediates NOTCH transcriptional control (Fig. 1C). The NICD is generated in response to receptor activation by ligand or Ca2⫹ chelation [28]. NICD was detected (shown in Fig. 1C) because the proteins were extracted from cells that had been maintained by dissociation with typsin-EDTA at passage, and the cells were harvested by typsin-EDTA dissociation prior to protein isolation. In addition, we easily detected the expression of NOTCH3 mRNA by RT-PCR, but we detected no or very low levels of NOTCH4 expression in the two cell lines we examined (Fig. 1D). Previous studies also found that mRNAs for NOTCH1–3 but not NOTCH4 are expressed in hESCs [13, 29]. We also find that presenilin-1 and nicastrin, two proteins that are key components of the ␥-secretase activity, are also expressed in www.StemCells.com
hESCs and are present in their active forms (Fig. 1C). An antibody that recognizes the loop of the C-terminal fragment of Presenilin-1 shows that the processed and active 20-kDa Cterminal fragment form is much more abundant than the uncleaved and inactive form in hESCs (Fig. 1C). Most of the nicastrin protein is present in its larger glycosylated and active form (Fig. 1C). Our results show that NOTCH receptor proteins, as well as key components required for the cleavage of NOTCH in response to ligand, are expressed in hESCs.
The NOTCH Pathway Can Be Activated in hESCs by Chelating Extracellular Cations It is well-established that NOTCH signaling is activated by treating cells with cation chelators [28]. We tested the ability of EDTA, a calcium/magnesium chelator and a component of cell dispersal reagents used in cell culture, to activate NOTCH. In these experiments, we monitored the levels of NICD with an antibody that specifically recognizes it [20, 30]. We compared the level of NICD in BGN1 cells that had been dispersed using collagenase plus trypsin-EDTA (Fig. 2A, ⫹EDTA lanes) with the level in BGN1 cells that had been dispersed by exposure to
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collagenase type IV without EDTA (⫺EDTA lanes). We detected ␥-secretase-cleaved NOTCH1 only in cells exposed to EDTA (Fig. 2A). This shows that NOTCH cleavage can be activated in hESCs when passaged by trypsin-EDTA. Treatment of the hESCs with a potent and specific ␥-secretase inhibitor prior to disrupting the cells with trypsin-EDTA blocked the production of the NICD fragment in the cells (Fig. 2B), indicating that the NOTCH activation resulted from intramembrane cleavage of NOTCH1 by ␥-secretase. Thus, an active ␥-secretase complex is present in the human ES cells and can generate NICD upon treatment with EDTA. We also tested whether cation chelation in hESCs could activate HES1, a known NOTCH target. We measured the level of endogenous HES1 transcripts in cells that had been exposed to EDTA or in cells treated with EDTA in the presence of the ␥-secretase inhibitor DAPT (Fig. 2C). EDTA exposure resulted in a fourfold induction of the endogenous HES1 gene in the hESCs (Fig. 2C; p ⬍ .001). This induction was reduced by half by the addition of DAPT (Fig. 2C; p ⬍ .05). Thus, blocking ␥-secretase blocks the induced upregulation of a NOTCH target gene. We also find that ␥-secretase inhibition does not reduce the expression of HES1 in cells not treated with EDTA (Fig. 2C), suggesting that NOTCH signaling via HES1 is not active in undifferentiated hESCs. The ability of EDTA to activate NOTCH signaling and induce the expression of the HES1 indicates that the NOTCH pathway can be activated by extracellular cation chelation in hESC cultures. To test whether NOTCH signaling is constitutively active in undifferentiated hESCs, we transiently transfected luciferase reporter vectors into BGN1 and H1. To avoid activating NOTCH, we used collagenase to passage the cells without exposure to EDTA. Two reporter vectors were used: one contains four copies of the consensus CBF1 binding site and is upregulated in response to NOTCH signaling, whereas the other is a control vector containing four mutated CBF1 binding sites [26]. We found that the wild-type and mutant CBF1-luciferase reporters were expressed at the same level after 2 days in the presence or absence of conditioned medium; however, transfection of the Notch NICD resulted in robust activation of the reporter (Fig. 2D). Control transfections confirmed that the NICD specifically activates the wild-type CBF reporter (Fig. 2E). The results show that although the pathway can be activated to induce NOTCH-specific gene expression, the canonical NOTCH signaling pathway is not active in hESCs within the limits of detection of our assays. Our results suggest that the NOTCH pathway in hESCs is normally quiescent but can be rapidly activated.
Inhibition of ␥-Secretase Reduces the Proportion of Differentiating Cells in hESC Cultures Our analysis indicates that NOTCH signaling can be activated in hESCs but normally remains inactive in undifferentiated cells. This suggests that NOTCH may not be involved in the maintenance of undifferentiated hESCs but may be required at some later point in differentiation. We tested this by inhibiting NOTCH signaling while propagating hESCs in conditions known to cause the accumulation of differentiated cells within the cultures. If NOTCH is required for the maintenance of undifferentiated hESCs, its inhibition by DAPT would lead to the rapid accumulation of SSEA4(⫺) cells and to changes in cell morphology. If it is required for the formation or maintenance of
Notch Signaling in Human ES Cells differentiated cell types within the cultures, then NOTCH inhibition would prevent or eliminate the accumulation of the differentiating SSEA4(⫺) cell population. To test the role of NOTCH in hESCs, we inhibited NOTCH cleavage by incubating cultures in the ␥-secretase inhibitor DAPT and measured the proportion of cells expressing NOTCH1 and SSEA4. To start the experiment with a relatively uniform cell population, we enriched for cells expressing high levels of SSEA4 by MACS sorting. The retained fraction was plated and subjected to the treatment conditions. Incubation with DAPT led to a statistically significant 27% reduction in total cell number relative to DMSO-treated controls (p ⫽ .0078; n ⫽ 8; Wilcoxon signed rank test). This reduction is due to a loss of cells expressing low or undetectable levels of SSEA-4 (Fig. 3). To determine the distribution of SSEA4 and NOTCH expression within the ␥-secretase inhibitor-treated cultures and controls, we measured the number of cells expressing NOTCH1 and/or SSEA4 by flow cytometry (Fig. 3A). The majority of cells in the parent culture (Fig. 3A, top left corner plot) express high levels of SSEA4 and NOTCH1 (FrA, 67.8%) indicative of the undifferentiated state of hESCs. A small portion expresses low levels of SSEA4 while expressing moderate levels of NOTCH1 (FrB, 28%). With DAPT treatment (Fig. 3A), the proportion of cells in the culture expressing low levels of SSEA4 is decreased, and the proportion expressing high levels of SSEA4 is increased compared with parent cultures and DMSOtreated or untreated cultures. In addition, compared with the parent culture, both lack of treatment and DMSO treatments decreased the proportion of SSEA4⫹ cells. Summary data for four independent experiments with two different hESC lines (BGN1 and BGN2) are shown as graphs in Figure 3B. An average of 30% of cells were in SSEA4low/NOTCH1⫹ fraction in the DMSO-treated cultures, whereas this was reduced by half with inhibitor treatment (p ⫽ .0286; n ⫽ 4; Mann-Whitney test). Therefore, inhibition of ␥-secretase significantly reduces the number and proportion of differentiating cells in hESC cultures.
Notch Signaling Is Activated in Differentiated Cells Derived from hESCs
The loss of differentiated cells from the ␥-secretase-treated cultures suggests that Notch signaling is active in the cells that have differentiated from the hESCs. To test this, we examined the expression of the NICD within spontaneously differentiating hESC cultures. In this case, we used cells that had been maintained exclusively by manual microdissection passaging. In manually passaged cultures, colony fragments sometimes do not attach immediately after being transferred to a new feeder layer. The fragments that are delayed in attachment form heterogeneous colonies containing undifferentiated and differentiated cell types. The differentiating cells within these colonies can be readily recognized by their columnar epithelial morphology. As the cells differentiate, they form tightly packed cell aggregates (Fig. 4B, 4C, black arrows). The tightly packed aggregates of differentiated cells (Fig. 4B, 4C, black arrows) clearly have a different appearance from the epithelial morphology of the undifferentiated cells in these cultures (Fig. 4C). After 5 days of differentiation, we found strikingly high levels of nuclear NICD staining in the cells within the differentiated cell aggregates of the partially differentiated hESC colonies (Fig. 4B, 4C). In undifferentiated cells, we could easily detect Notch receptor expression (Fig. 1A), as well as Oct4 expression (Fig. 4D), but
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Figure 3. Inhibition of ␥-secretase reduces the proportion of differentiated cells in human embryonic stem cell (hESC) cultures. (A): Fluorescence-activated cell sorting (FACS) analysis of cells labeled with antibodies to NOTCH1 and SSEA4. The cell populations are resolved into FrA and FrB. FACS analysis of three independent cultures from the same experiment is shown. Samples were untreated, incubated in DMSO, or incubated in DAPT dissolved in DMSO as indicated. Control plots are shown in the bottom row. (B): A summary of the ␥-secretase treatment results from four independent experiments with the two hESC lines, BGN1 and BGN2. Each graph shows cell number (top row) or percentage of total cells (bottom row) in each of the fractions (FrB and FrA). The white bars show the results for DMSOtreated control cultures, whereas the shaded bars show the results for DAPT-treated cultures. Significance was determined by Mann-Whitney tests. Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycine t-butyl ester; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; FrA, NOTCH⫹SSEA4⫹ fraction; FrB, NOTCH ⫹SSEA4low/⫺ fraction; PE, phycoerythrin; SSEA, stage-specific embryonic antigen.
we did not detect NICD (Fig. 4B, 4C). The presence of high levels of nuclear NICD in differentiated cells suggests that Notch signaling becomes active in the differentiating cells derived from hESCs.
DISCUSSION Our results show that NOTCH signaling can be activated in undifferentiated hESCs. We found that mRNAs encoding NOTCH 1, 2, and 3, as well as the NOTCH1 and NOTCH2 proteins, are present in hESCs. In addition, key components of the ␥-secretase activity are also present in their processed and active forms. Furthermore, www.StemCells.com
transcription of HES1, a NOTCH target, is induced in hESCs after NOTCH activation. Our analysis shows that NOTCH signaling is normally inactive in undifferentiated hESCs. We found that NOTCH signaling appears to be greatly upregulated as the cells differentiate because the differentiating cells have high levels of NICD present in their nuclei. This observation is consistent with our finding that incubation of hESC cultures in a ␥-secretase inhibitor results in a reduction in the proportion of differentiated cells in the cultures. The results are consistent with a role for NOTCH signaling in the maintenance of differentiating cell types in hESC cultures.
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Figure 4. Notch signaling is activated upon spontaneous differentiation of human embryonic stem cells (hESCs). BG01 hESCs at passage 30 were microdissected and plated on mouse embryonic fibroblasts (MEFs). Several colonies that delayed attachment to the feeder layer produced spontaneously differentiating colonies that contained aggregates of differentiating cells and regions of undifferentiated epithelial hESCs (B–D). After 5 days of differentiation, the cultures were stained with the Notch intracellular domain (NICD)-specific Val1744 antibody and detected with horseradish peroxidase-conjugated secondary antibodies (B, C). A reaction omitting primary antibody is shown as a control (A). Note intense NICD staining in aggregates of differentiated cells (B, C, black arrows). The undifferentiated cells within these colonies exhibited the characteristic epithelial hESC morphology and did not contain detectable levels of NICD (C). The undifferentiated hESCs expressed high levels of Oct4 (D, white arrow). (B): Box indicates the region shown at higher magnification in (C). Scale bars ⫽ 200 m.
The requirement for NOTCH signaling for the survival of differentiated cells in hESC cultures is consistent with the known requirement for NOTCH in the formation and maintenance of differentiating cell types in mouse embryos. Although the mRNAs for Notch pathway components are present in mouse ES cells and in mouse embryos during preimplantation and pregastrula stages [31, 32], Notch signaling is not required in mouse ES cells or during the early stages of mouse embryogenesis [33–35]. A requirement for Notch is first detected at the earliest stages of organogenesis at E8.5–9.5 in Pofut1, CBF1, or presenilin (PS) mutant mice [35, 36]. Detailed analysis of the developing central nervous system in PS1⫺/⫺ and PS1⫺/⫺; PS2⫹/⫺ mice indicates a greatly reduced self-renewing neural stem cell population, suggesting that Notch is required for the maintenance of neural stem cell populations [36]. The results of genetic analysis in mouse ES cells and mice suggest that Notch signaling components are present but inactive prior to the emergence of tissue-specific stem cell populations during and after gastrulation. Our results suggest that the timing of NOTCH function during human embryonic differentiation is similar to that found in the mouse system.
REFERENCES 1
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.
2
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.
From a practical standpoint, the accumulation of differentiating cells in hESC cultures interferes with the maintenance of undifferentiated cells and with controlled differentiation. The cell types that can accumulate in hESC cultures are a highly heterogeneous population [9]. The extracellular factors produced by such cells can stimulate the differentiation of the ES cells, leading to increasing heterogeneity within the cultures and the eventual loss of the hESCs. The ability of accumulating differentiated cells to induce the differentiation of the remaining undifferentiated ES cells has been shown previously for mouse embryonic stem cells [37, 38]. Our results indicate that ␥-secretase inhibitors reduce the accumulation of differentiating cells within the hESC and may be extremely useful for the maintenance of undifferentiated hESC lines. It is important to note that our data also show that NOTCH signaling can be activated in hESCs by reagents used for dissociating cells. We and others have noted that differentiated cells, as well as aneuploid undifferentiated hESCs, accumulate in cultures that are maintained over many cell passages (more than 10) using complete cell dissociation with EDTA-containing solutions [9, 39, 40]. As a result, manual microdissection passaging has been proposed as a method for the maintenance of undifferentiated hESC cultures. We suggest that the repeated activation of NOTCH signaling by cation chelation during cell dissociation may provide a transient survival signal to differentiated cell types. Further studies of NOTCH signaling, as well as the activity of other pathways in hESCs cultured in a variety of conditions, may lead to the development of methods that support routine cell dissociation passaging of most if not all hESC lines. This is a highly desirable goal because manual microdissection passaging is a barrier to the production of the cell numbers required for routine experimentation. A greater understanding of the signals that control the self-renewal, differentiation, and proliferation of hESCs should lead to culture systems that can generate the large numbers of normal and undifferentiated cells required for routine experimentation and cell therapies.
ACKNOWLEDGMENTS We thank Dr. Ali Hemmati-Brivanlou for discussions, Drs. S. Diane Hayward and Raphael Kopan for plasmids, and Dr. Nancy Manley for reading the manuscript. The monoclonal antibodies bTAN20 (NOTCH1) and C651.6bHN (NOTCH2) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). This work was supported by NIH Award MH064794 (to B.G.C.) and by BresaGen Inc.
DISCLOSURES B.C. has acted as a consultant for and has received financial support from BresaGen within the last 2 years.
3
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.
4
Vallier L, Reynolds D, Pedersen RA. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol 2004;275:403– 421.
Noggle, Weiler, Condie
5
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.
6
James D, Levine AJ, Besser D et al. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 2005;132:1273–1282.
7
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360 –380.
8
Bieberich E, Silva J, Wang G et al. Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants. J Cell Biol 2004;167:723–734.
9
Mitalipova MM, Rao RR, Hoyer DM et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 2005;23:19 – 20.
10 Lai EC. Notch signaling: Control of cell communication and cell fate. Development 2004;131:965–973. 11 Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: Cell fate control and signal integration in development. Science 1999;284:770 – 776. 12 Kadesch T. Notch signaling: The demise of elegant simplicity. Curr Opin Genet Dev 2004;14:506 –512. 13 Walsh J, Andrews PW. Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell. APMIS 2003;111:197–210; discussion 210 –191. 14 Schulz TC, Noggle SA, Palmarini GM et al. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. STEM CELLS 2004;22:1218 –1238. 15 Kornilova AY, Das C, Wolfe MS. Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications. J Biol Chem 2003; 278:16470 –16473. 16 Kornilova AY, Bihel F, Das C et al. The initial substrate-binding site of gamma-secretase is located on presenilin near the active site. Proc Natl Acad Sci U S A 2005;102:3230 –3235. 17 Micchelli CA, Esler WP, Kimberly WT et al. Gamma-secretase/presenilin inhibitors for Alzheimer’s disease phenocopy Notch mutations in Drosophila. FASEB J 2003;17:79 – 81. 18 Geling A, Steiner H, Willem M et al. A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 2002;3:688 – 694. 19 Yeh JR, Crews CM. Chemical genetics: Adding to the developmental biology toolbox. Dev Cell 2003;5:11–19. 20 Cheng HT, Miner JH, Lin M et al. Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney. Development 2003;130:5031–5042.
1653
23 De Smedt M, Hoebeke I, Reynvoet K et al. Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment. Blood 2005;106: 3498 –3506. 24 Soriano S, Kang DE, Fu M et al. Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing. J Cell Biol 2001;152:785–794. 25 Koo EH, Kopan R. Potential role of presenilin-regulated signaling pathways in sporadic neurodegeneration. Nat Med 2004;10(suppl):S26 –S33. 26 Hsieh JJ, Henkel T, Salmon P et al. Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol Cell Biol 1996;16:952–959. 27 Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974. 28 Rand MD, Grimm LM, Artavanis-Tsakonas S et al. Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 2000;20:1825–1835. 29 Brandenberger R, Khrebtukova I, Thies RS et al. MPSS profiling of human embryonic stem cells. BMC Dev Biol 2004;4:10. 30 Phiel CJ, Wilson CA, Lee VM et al. GSK-3alpha regulates production of Alzheimer’s disease amyloid-beta peptides. Nature 2003;423:435– 439. 31 Cormier S, Vandormael-Pournin S, Babinet C et al. Developmental expression of the Notch signaling pathway genes during mouse preimplantation development. Gene Expr Patterns 2004;4:713–717. 32 Wang QT, Piotrowska K, Ciemerych MA et al. A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 2004;6:133–144. 33 Shi S, Stahl M, Lu L et al. Canonical notch signaling is dispensable for early cell fate specifications in mammals. Mol Cell Biol 2005;25:9503– 9508. 34 Herreman A, Serneels L, Annaert W et al. Total inactivation of gammasecretase activity in presenilin-deficient embryonic stem cells. Nat Cell Biol 2000;2:461– 462. 35 Donoviel DB, Hadjantonakis AK, Ikeda M et al. Mice lacking both presenilin genes exhibit early embryonic patterning defects. Genes Dev 1999;13:2801–2810. 36 Hitoshi S, Alexson T, Tropepe V et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002;16:846 – 858. 37 McWhir J, Schnieke AE, Ansell R et al. Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background. Nat Genet 1996;14: 223–226. 38 Mountford P, Nichols J, Zevnik B et al. Maintenance of pluripotential embryonic stem cells by stem cell selection. Reprod Fertil Dev 1998;10: 527–533.
21 van den Brandt J, Voss K, Schott M et al. Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineage. Eur J Immunol 2004;34:1405–1413.
39 Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004; 22:53–54.
22 Timmerman LA, Grego-Bessa J, Raya A et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev 2004;18:99 –115.
40 Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382; author reply 382.
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