Embryoid Body Formation of Human Amniotic Fluid Stem Cells Depends

Oncogene (2010) 29, 966–977 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Embryoid body formation of human amniotic ﬂuid stem cells depends on mTOR

A Valli1, M Rosner1, C Fuchs1, N Siegel1, CE Bishop2, H Dolznig1,UMa¨del3, W Feichtinger3, A Atala2 and M Hengstschla¨ger1

1Department of Medical Genetics, Medical University of Vienna, Vienna, Austria; 2Department of Urology and Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA and 3Wunschbabyzentrum, Lainzer Strabe 6, Vienna, Austria

Human amniotic fluid stem cells (hAFSCs) harbor high directly in somatic cells (Pennings, 2007; Aboushwareb and proliferative capacity and high differentiation potential Atala, 2008; Maherali and Hochedlinger, 2008). and do not raise the ethical concerns associated with Since the description of cells within the human human embryonic stem cells. The formation of three- amniotic fluid expressing the transcription factor Oct4, dimensional aggregates known as embryoid bodies (EBs) a marker for pluripotent human stem cells (Prusa et al., is the principal step in the differentiation of pluripotent 2003), a variety of different stem cell populations have embryonic stem cells. Using c-Kit-positive hAFSC lines, been described to exist in human amniotic fluid. AFSCs we show here that these stem cells harbor the potential to have been shown to harbor the potential for osteogenic, form EBs. As part of the two kinase complexes, mTORC1 chondrogenic, adipogenic, renal, hematopoietic or neu- and mTORC2, mammalian target of rapamycin (mTOR) rogenic differentiation (in ‘t Anker et al., 2003; Prusa is the key component of an important signaling pathway, et al., 2004; Tsai et al., 2004, 2006; Karlmark et al., 2005; which is involved in the regulation of cell proliferation, Bossolasco et al., 2006; Kim et al., 2007; Kolambkar growth, tumor development and differentiation. Blocking et al., 2007; Perin et al., 2007; Ditadi et al., 2009). intracellular mTOR activity through the inhibitor rapa- Importantly, using immunoselected CD117 (c-Kit)-posi- mycin or through specific small interfering RNA tive hAFSCs it has been shown that, descending from a approaches revealed hAFSC EB formation to depend on single cell, differentiation along adipogenic, osteogenic, mTORC1 and mTORC2. These findings demonstrate myogenic, endothelial, neurogenic and hepatic lineages hAFSCs to be a new and powerful biological system to could be induced. These hAFSCs can be expanded in recapitulate the three-dimensional and tissue level con- culture as stable lines with high proliferative capacity texts of in vivo development and identify the mTOR and do not induce tumor formation in mice (De Coppi pathway to be essential for this process. et al., 2007). hAFSCs might have advantages (such as a Oncogene (2010) 29, 966–977; doi:10.1038/onc.2009.405; higher proliferation rate and a higher differentiation published online 23 November 2009 potential) over other adult stem cell types. They even might have advantages (such as a lower risk for tumor Keywords: amniotic fluid; stem cell; EB; mTOR development and no ethical concerns) over embryonic stem cells. In addition, hAFSCs might allow to study specific differentiation processes under normal and pathological conditions with the aim to clarify how the Introduction underlying molecular mechanisms are deregulated in cells harboring natural occurring molecular alterations It was always assumed that both the differentiation being causatively involved in the development of specific potential and proliferative capacity of adult stem cells are human genetic diseases (reviewed in Siegel et al., 2007; limited compared with embryonic stem cells. However, Perin et al., 2008; Cananzi et al., 2009). However, adult stem cells do not raise ethical concerns and harbor a embryonic stem cells, when cultured without antidiffer- low risk of tumor development. Many different studies entiation factors, can spontaneously form three-dimen- havebeeninitiatedtosearchfor alternative human sources sional multicellular aggregates called embryoid bodies for stem cells harboring the potential to differentiate into (EBs). This cell biological process allows the recapitula- specific lineages, including the induction of pluripotency tion and investigation of the three-dimensional and tissue level contexts of many cell differentiation phenom- ena during early mammalian embryogenesis (Itskovitz- Correspondence: Professor M Hengstschla¨ger, Department of Medical Eldor et al., 2000; Koike et al., 2007; Ungrin et al., 2008). Genetics, Medical University of Vienna, Wa¨hringer Gu¨rtel 18–20, The question whether hAFSCs harbor the potential to Vienna 1090, Austria. form EBs remained elusive so far. E-mail: [email protected] Received 18 May 2009; revised 4 September 2009; accepted 15 October The mammalian target of rapamycin (mTOR) kinase 2009; published online 23 November 2009 has a central role in the regulation of cell proliferation, EB formation of hAFSCs depends on mTOR A Valli et al 967 growth, tumor development and many cell differentiation (medium 2; Narita et al., 1996; Kurosawa, 2007) processes. In mammalian cells two different mTOR (Table 1). In this study two standard commonly used containing complexes, namely mTORC1 and mTORC2, approaches, the hanging drop method and cultivation in have been identified. mTORC1 contains the mTOR suspension in low-adherence 96-well plates, were used to protein, raptor and mLST8. mTORC2 consists of induce EB formation of hAFSCs. mTOR, mLST8, rictor and sin1. mTORC1 is involved We have cultivated CD117/2, Q1 and CB3 cells, in the regulation of mRNA translation, for example, resuspended in medium1 or medium 2 for 4 days in through its potential to phosphorylate and activate one hanging drop cultures. Thereafter, the formed EBs were of its major targets, the kinase p70S6K, at T389 to transferred on 0.1% gelatin-coated tissue culture dishes activate the ribosomal protein S6 through phosphoryla- (Figure 2). The incidence of EB formation (given as tion at S240/244. mTORC2 phosphorylates Akt at S473, percentage of the number of attached EBs recovered which, in conjunction with the PDK1-mediated phos- from 15 hanging drops seeded) was investigated in three phorylation at T308, drives full activation of Akt. independent experiments. Each independent experiment Upstream of mTOR, activated receptor tyrosine kinases was carried out by seeding 15 hanging drops in activate the kinase PI3K through, for example, IRS1, triplicate. Compared with the other two cell lines, regulating the potential of PDK1 to phosphorylate Akt. CD117/2 cells exhibited a lower EB formation incidence, Akt-mediated phosphorylation downregulates the which might be due to the fact that these hAFSCs do GTPase-activating potential of tuberin (TSC2) toward not necessarily represent a clonal cell population Rheb, which is a potent regulator of mTORC1. The (Table 2). In summary, EB formation was observed in tuberin/hamartin complex can also associate with and every experiment using three different hAFSC lines and positively regulate mTORC2 (Wullschleger et al., 2006; two different cultivation media. Dann et al., 2007; Guertin and Sabatini, 2007; Yang and Guan, 2007). Marker expression in EBs derived from hAFSCs Here we demonstrate hAFSCs to harbor the potential The Oct4 transcription factor is a well-known stem cell to form EBs and we identify mTOR as a major regulator marker for pluripotency (Pesce and Scho¨ler, 2001). of this cell biological process. Our findings provide Oct4-positive cells were detected in human amniotic important additional evidence that hAFSCs indeed fluid (Prusa et al., 2003) and the CD117-positive hAFSC represent a powerful tool for human developmental lines have been shown to also express Oct4 (De Coppi biology and allow new insights into the underlying et al., 2007). Performing reverse transcriptase–PCR molecular mechanism of EB formation. experiments, we have confirmed hAFSCs to be Oct4- positive. We found hAFSCs to also express the self- renewal (human embryonic) stem cell marker nodal (Besser, 2004) (Figure 3). We observed EB formation of Results hAFSCs to be accompanied by a decrease of nodal and Oct-4 expression and by induction of the differentiation Characterization of CD117-positive hAFSC lines markers Pax 6 (ectodermal), Flk1 (endothelial), E- In addition to the earlier established clonal hAFSC line, cadherin (epithelial), GATA4 (endodermal), T (brachy- Q1, (De Coppi et al., 2007) we generated two new ury; mesodermal) and HBE1 (mesodermal) (Figure 3), CD117-positive hAFSC lines, namely CB3 and CD117/2, which is in agreement with the observations on EB from native human amniotic fluid cell samples using the formation of embryonic stem cells (Narita et al., 1996; previously described protocol for immunoselection Itskovitz-Eldor et al., 2000; Yamashita et al., 2000; through magnetic cell sorting. Besser, 2004; Ng et al., 2005; Koike et al., 2007; The morphological appearance and cell size of CD117/2 Kurosawa, 2007; Gualandris et al., 2008; Kim et al., cells, established without prior minimal dilution, was very 2008; Lim et al., 2008; Ungrin et al., 2008). comparable with Q1 and CB3 hAFSCs, descending from Confocal microscopy of immunocytochemical inves- one single cell on minimal dilution. All three cell lines grow tigations further proved hAFSCs to be positive for Oct- without the need of feeder layers, without evidence for 4 and negative for Flk1. In case of E-cadherin, the spontaneous differentiation, with stable normal karyo- expression level was between extremely low and types, with slight differences in the amount of cells in the undetectable through immunocytochemistry (Figure 4). different cell cycle phases and without significant appear- On induction of EB formation in suspension, we ance of apoptotic sub-G1 cells (Figure 1). In summary, detected the characteristic formation of a distinct these data again prove that the described magnetic cell peripheral layer on the outer surface of the EBs, sorting approach is a reproducible method to generate including cells that express laminin or a-fetoprotein. In genomically stable CD117-positive hAFSC lines. addition, these confocal analyses showed specific cells within the EBs to express the endothelial marker Flk1, EB formation of hAFSCs the epithelial marker E-cadherin or the ectodermal To answer the question whether hAFSCs can form EBs, marker nestin (Figure 5). Here it is important to note we decided to use two different growth media, one that that the vascular endothelial growth factor receptor 2, has already been used earlier to cultivate hAFSCs Flk1, is known to be localized to both the cytoplasm and (medium 1; De Coppi et al., 2007) and another one, nucleus, depending on its phosphorylation status which is commonly used for embryonic stem cell growth (Yamashita et al., 2000; Blazquez et al., 2006).

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 968 CD117/2 Q1 CB3 Karyotype: 46,XY Karyotype: 46,XY Karyotype: 46,XX

% G0/G1: 64.5±1.0 2400 % G0/G1: 46.8±0.7 % G0/G1: 67.2±2.2 2800 % S: 34.8±0.6 3200 % S: 24.2±1.8 % S: 30.9±0.7 1800 % G2/M: 18.4±0.9 % G2/M: 4.6±0.5 2400 % G2/M: 8.6±0.4 2100

1200 Counts 1800 Counts 1400 Counts 1200 800 700

0 0 0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 DNA content DNA content DNA content % Sub-G1: 0.64 ±0.05 % Sub-G1: 0.62 ±0.06 % Sub-G1: 0.74 ±0.03

400 CD117/2 2000 CB3 320 Q1 IMR-90 1500 240

1000 Counts 160 Femtoliter 500 80

0 0 0 200 400 600 800 1000 CD117/2 Q1 CB3 Forward scatter Figure 1 Biological characterization of the used human amniotic fluid stem cell (hAFSC) lines. (a) Cytogenetical analyses revealed normal and stable karyotypes for the used hAFSC lines, CD117/2, Q1 and CB3 (data not shown). Logarithmically growing cells were microscopically analysed for cell morphology and cytofluorometrically analysed for DNA content. For the latter, the percentage (±s.d.) of cells in G0/G1, S and G2/M phases of the cell cycle and the percentage (±s.d.) of apoptotic cells with sub-G1 DNA is presented. (b) The cell size of the three hAFSC lines was compared with the cell size of nonimmortalized nontransformed primary human IMR-90 fibroblasts through forward scatter analyses. (c) Logarithmically growing CD117/2, Q1 and CB3 cells were analysed for cell size in femtoliter on the Casy Cell Counter.

Table 1 The cell culture media used Medium 1 Medium 2

a-MEM (Gibco-41061; Invitrogen, Carlsbad, CA, USA) DMEM (Gibco-11960z) Penicillin 0.03 mg/ml Penicillin 0.03 mg/ml Streptomycin 0.05 mg/ml Streptomycin 0.05 mg/ml Chang B 18% 2-mercaptoethanol 0.1 mM Chang C 2% Glutamine 2 mM Fetal bovine serum (HyClone-30070.03; Waltham, MA, USA) 15% Fetal bovine serum (Sigma-F7524) 15%

Abbreviations: DMEM, Dulbecco’s modiﬁed Eagle’s medium; a-MEM, a-Minimal Essential Medium.

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 969 CD117/2 Q1 CB3

Day 5 Medium 1

Day 15

Day 5 Medium 2

Day 15

Figure 2 Embryoid body (EB) formation of CD117/2, Q1 and CB3 cells was induced using the hanging drop method as described in the Materials and methods section. For the two different media used in these experiments compare Table 1. Pictures of representative of EBs detected on day 5 (4 days in hanging drop and 1 day after attachment in cell culture) in medium 1 (a–c), on day 15 (4 days in hanging drop and 11 days after attachment in cell culture) in medium 1 (d–f), on day 5 in medium 2 (g–i) and on day 15 in medium 2 (j–l) are presented. Scale bar: 100 mm.

Table 2 Incidence of EB formation in the different hAFSC lines Medium CD117/2 Q1 CB3

1 212 12

EB formation 38%±10 18%±10 69%±3 40%±13 76%±10 82%±4 (average±s.d.)

Abbreviations: EB, embryoid body; hAFSC, human amniotic ﬂuid stem cell.

The conclusion that different cells with different EB formation of hAFSCs depends on mTOR differentiation status are part of hAFSC-derived EBs Conﬂicting results from studies in murine models have is supported by our observation that, within one EB, been published regarding the role of mTOR for the cells positive and negative for speciﬁc differentiation development of embryonic and extraembryonic tissues markers exist (Figure 5). (Gangloff et al., 2004; Shiota et al., 2006; Sampath et al.,

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 970 2008). To test the role of mTOR for EB formation of The incidence of EB formation of hAFSCs (given as hAFSCs, we made use of the widely used mTOR percentage of the number of attached EBs recovered inhibitor rapamycin. First, we found that blocking from 15 hanging drops seeded) was significantly mTOR activity significantly affected the cell cycle diminished on negatively regulating mTOR through regulation and proliferation rate of hAFSCs without rapamycin (Figure 7c; Po0.05). In addition, rapamycin inducing apoptosis (Figure 6). treatment caused a pronounced effect on EB size Western blot analyses revealed that rapamycin treat- (Figures 7d and e; Po0.05). Taken together, these ment triggered a block of mTOR-regulated S6 phos- findings show for the first time that mTOR has a major phorylation in hAFSCs (Figures 6c, 7a). In addition, role in the EB formation process of hAFSCs. FSC analyses of cells of the induced EBs showed that rapamycin induced a downregulation of hAFSC size (Figure 7b), which is in perfect agreement with The role of mTORC1 and mTORC2 for EB formation previously reported effects of mTOR on cell size of hAFSCs regulation (compare, for example, Sarbassov et al., In mammalian cells, two mTOR-containing complexes, 2006; Rosner and Hengstschla¨ger, 2008). namely mTORC1 and mTORC2, with different substrate specificities have been described. Rapamycin bound to the protein FKBP12 generates a drug–receptor complex that binds and inhibits mTORC1. As FKBP12- rapamycin does not bind to preformed mTORC2, rapamycin was originally thought to only inhibit mTORC1 (Wullschleger et al., 2006; Guertin and Sabatini, 2007; Yang and Guan, 2007). However, recently it was shown that long-term rapamycin treatment also suppresses the function of mTORC2 and that this property is important for the effects of this drug (Sarbassov et al., 2006; Rosner and Hengstschla¨ger, 2008). Accordingly, the fact that rapamycin affects the EB formation process of hAFSCs described above made it interesting to further investigate the specific roles of mTORC1 and mTORC2. We carried out small interfering RNA (siRNA) experiments to downregulate the endogenous protein levels of either the mTORC1- Figure 3 Marker expression before and after embryoid body (EB) formation of human amniotic fluid stem cells (hAFSCs). Loga- specific component raptor or of the mTORC2-specific rithmically growing CB3 cells (C, control) have been induced to component rictor in hAFSCs. Western blot analyses form EBs (day 15). The expression of Pax6 mRNA, HBE1 mRNA, revealed that raptor-specific siRNA treatment caused a nodal mRNA, Flk1 mRNA, GATA4 mRNA, T (brachyury) pronounced downregulation of endogenous raptor mRNA, Oct4 mRNA and E-cadherin mRNA was analysed using reverse transcriptase–PCR. b-actin mRNA expression was co- protein levels without effects on rictor and vice versa analysed as a control. All primers used have been shown to be (Figure 8a). Here it is important to note that the siRNA- specific earlier (see Materials and methods). mediated downregulation of these proteins in hAFSCs

Figure 4 Marker expression in CB3 human amniotic ﬂuid stem cells (hAFSCs). CB3 cells were grown on slides, ﬁxed, permeabilized and incubated with antibodies against the indicated proteins (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). A control experiment, in which the primary antibody has been omitted, is presented. Scale bars: 20 mm.

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 971

Figure 5 Marker analyses in CB3 human amniotic fluid stem cells (hAFSCs)-derived embryoid bodies (EBs). CB3 cells were induced to form EBs in suspension using low-adherence 96-well plates, fixed after 6 days of culture, permeabilized and stained for several differentiation markers (green). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). (a) a-Fetoprotein (aFP) and laminin (Lam) expression in EBs. EBs solely stained with the fluorescent-labelled secondary antibody served as negative control (Control). A phase-contrast microscopic image is included (right). (b) Laminin is expressed in the most outer layer of cells (white arrows). Cells beneath the outer structures are negative for laminin (asterisks). A distinct outer layer was also seen using phase-contrast microscopy (right, black arrows). (c) Flk1 expression was detectable in a subset of cells (white arrows). Asterisks indicate cells that are negative for Flk1. (d) E-cadherin (E-cad) staining and corresponding phase contrast image. (e) Nestin staining and corresponding phase contrast image. Scale bars: 20 mm. remained stable over the time period of the EB statistical significance (Figure 8b). However, both formation experiments performed here (data not raptor-specific and rictor-specific siRNA treatment shown). Raptor-specific siRNA treatment triggered a caused pronounced effects on EB size (Figure 8c, d; downregulation of the amount of phosphorylated S6 Po0.05). These data confirm that mTOR is a major S240/244, which is a substrate of the mTORC1- regulator of EB formation of hAFSCs. However, these regulated p70S6 Kinase, without negative effects on results also highlight that there might be differences in the amount of phosphorylated Akt S473, which is a the role of mTORC1 and mTORC2 for this process. substrate of mTORC2. However, although, as expected, rictor-specific siRNA treatment caused a strong reduc- tion of endogenous phospho-Akt S473 levels, it also seemed to affect the S6 S240/244 phosphorylation levels Discussion (Figure 8a). The incidence of EB formation of hAFSCs (given as Human amniotic fluid stem cells are on the way to percentage of the number of attached EBs recovered become an important source for both basic science and from 15 hanging drops seeded) was significantly regenerative medicine. Pluripotentiality and a high diminished on treatment with raptor-specific siRNAs proliferation rate are their advantages compared with (Figure 8b; Po0.05). Interestingly, rictor-specific siR- adult stem cells. Accessibility, no ethical concerns and NAs did not affect the incidence of EB formation with the fact that they do not produce teratomas when

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 972

Figure 6 Rapamycin-mediated effects on cell cycle progression and proliferation of human amniotic fluid stem cell (hAFSC) lines. (a) Logarithmically growing CB3 and Q1 cells were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100 nM for 24 h or with DMSO as a control, stained with propidiumiodide and cytofluorometrically analysed for DNA distribution. Representative DNA profiles are presented. (b) In addition, the percentage (±s.d.) of so treated cells in G0/G1, S and G2/M phase of the cell cycle is indicated. Nontransformed, nonimmortalized IMR-90 lung fibroblasts were analysed in parallel. (c) Cells as described above were treated with rapamycin for a total of 5 days and analysed for proliferation rates by counting cells at indicated time points (d1, day 1, represents cells collected after 24 h rapamycin treatment). Cell numbers are given as a percentage (±s. d.) relative to the number of cells seeded, set as 100%. At the final day 5, cells were additionally investigated for the phosphorylation status of the mTOR downstream effector ribosomal protein S6 by immunoblotting (upper right panel). (d) Besides, CB3, Q1 and IMR-90 cells analysed in (a) were cytofluorometrically investigated for the percentage (±s.d.) of apoptotic cells with sub-G1 DNA content.

transplanted to animals could be their advantages when the question whether these stem cell types merely allow compared with embryonic stem cells (Trounson, 2007; to investigate different specific differentiation processes Perin et al., 2008; Cananzi et al., 2009). In addition, of individual cells, or are also usable for studies of the comparable with induced pluripotent stem cells, according three-dimensional and tissue-level contexts. hAFSCs could be a useful source for the generation of Especially, so far, the potential to form EBs has not disease specific human stem cell lines providing an been proven for hAFSCs. optimal biological model to recapitulate normal and When cultured in suspension, under conditions in pathological differentiation processes in vitro. In the which they are unable to attach to the surface of culture past, tumor cell lines or transformed derivatives of dishes, without antidifferentiation factors and without native tissues from diseased patients represented the contact with feeder cells, embryonic stem cells harbor most commonly used biological systems for such the potential to form such three-dimensional aggregates, research studies. Nowadays, it has already been shown named EBs (Narita et al., 1996; Itskovitz-Eldor et al., to be possible to establish specific induced pluripotent 2000; Kurosawa, 2007; Ungrin et al., 2008). Here it is stem cells from patients diagnosed with different important to note that compared with mouse EBs, diseases (Dimos et al., 2008; Maherali and Hochedlin- human EB formation and the accompanied differentia- ger, 2008; Park et al., 2008). Worldwide, amniocenteses tion is known to be much more chaotic and disorganized are performed for prenatal genetic diagnosis of mono- with wide variability both between and within individual genetic diseases. It could be a promising strategy of EBs. However, three criteria must be fulfilled to define highest value for basic research to establish hAFSCs the potential of human stem cells to form EBs (Narita derived from a wide variety of pregnancies with specific et al., 1996; Itskovitz-Eldor et al., 2000; Koike et al., genetic aberrations. And compared with induced plur- 2007; Kurosawa, 2007; Kim et al., 2008; Ungrin et al., ipotent stem cells, hAFSCs do not need to undergo prior 2008): (1) It must be possible to reproducibly induce EB processes of inducing stem cell properties (Siegel et al., formation right under the growth conditions described 2007). However, the question whether induced pluripo- above. Performing many independent experiments using tent stem cells and hAFSCs harbor pluripotentiality of different approaches, we demonstrate in this report that the same level as embryonic stem cells is still under hAFSCs grown under such conditions can indeed form investigation. In addition, it is of importance to answer EBs. (2) EB formation does not only represent a change

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 973

Figure 7 Embryoid body (EB) formation of human amniotic fluid stem cell (hAFSC) depends on mammalian target of rapamycin (mTOR). (a) CB3 stem cells logarithmically growing in medium 1 (Table 1) were incubated with and without the mTOR inhibitor rapamycin at a final concentration of 100 nM and total protein lysates were prepared. These protein extracts were used to study the amount of phosphorylated S6 S240/244, which is a substrate of the mTOR-regulated p70S6 Kinase, and total S6 through western blot analyses. Detection of a-tubulin was included to prove equal loading of lysates. (b) CB3 cells with and without incubation with rapamycin were induced to form EBs in medium 1. EB cells were analysed for cell size through forward scatter determination of the flow cytometer. (c) The incidence of EB formation of CB3 cells on day 15 was investigated in three independent experiments. Each independent experiment was carried out by seeding 15 hanging drops in triplicate. The incidence is given as percentage (±s.d.) of the number of attached EBs recovered from 15 hanging drops seeded. (d) Representative pictures of CB3-derived EBs on day 15 in medium 1 with and without treatment with rapamycin. (e) The size of the EBs obtained in the experiments described in (c) and (d) was determined using the cell’D software from Olympus and is given in mm2 (±s.d.). in growth characteristics, but must rather be accom- during differentiation of murine embryonic stem cells panied by specific differentiation processes. As well into EBs has been profiled. It was reported that a variety described for EBs derived from human or mouse of translational regulators, including mTOR, control embryonic stem cells, EB formation of hAFSCs is global and selective protein synthesis during this process accompanied by a decrease of nodal and Oct-4 expres- and that translational regulation may be an important sion and by induction of the differentiation markers Pax quality control for self-renewal and choice of fate in 6 (ectodermal), nestin (ectodermal), Flk1 (endothelial), murine embryonic stem cells (Sampath et al., 2008). E-cadherin (epithelial), GATA4 (endodermal), T (bra- However, until now, the question whether mTOR chyury; mesodermal) and HBE1 (mesodermal). We function is essential for the development of murine or additionally detected the characteristic formation of a human EBs remained elusive. In this study, we show distinct peripheral layer on the outer surface of the EBs, such a role of mTOR for EB formation of hAFSCs. We including cells that express laminin or a-fetoprotein. (3) found the mTOR inhibitor rapamycin to significantly Finally, it was important to prove that different cells diminish both the incidence of EB formation of hAFSCs with different differentiation status are part of the and the size of these aggregates. generated EBs formed from one clonal hAFSC type. In In mammalian cells, two different mTOR complexes summary, to our knowledge this is the first demonstra- with different substrate specificities exist. mTORC1- tion that hAFSCs can form EBs and therefore can be containing raptor is involved in the regulation of used for investigations of the three-dimensional and mRNA translation through its potential to phosphor- tissue-level contexts of cell differentiation processes. ylate the p70S6 Kinase to activate the ribosomal protein The mTOR kinase is well known to have a central role S6 by phosphorylation at S240/244. mTORC2 contains in the regulation of many cell differentiation processes. rictor and phosphorylates Akt at S473, which, in However, conflicting results from studies in murine conjunction with the PDK1-mediated phosphorylation models have been published regarding the role of mTOR at T308, drives full activation of Akt (Wullschleger for the development of embryonic and extraembryonic et al., 2006; Dann et al., 2007; Guertin and Sabatini, tissues (Gangloff et al., 2004; Shiota et al., 2006; 2007; Yang and Guan, 2007). Recently it was shown Wullschleger et al., 2006; Guertin and Sabatini, 2007; that rapamycin suppresses the function of both Yang and Guan, 2007; Rosner et al., 2008). Very mTORC1 and mTORC2 (Sarbassov et al., 2006; Rosner recently, integrating transcriptome analysis with global and Hengstschla¨ger, 2008). Accordingly, our demon- assessment of ribosome loading, the gene expression stration that rapamycin affects the EB formation

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 974

Figure 8 Embryoid body (EB) formation of human amniotic fluid stem cell (hAFSC) depends on mTORC1 and mTORC2. (a) CB3 stem cells were transfected with unspecific control small interfering RNAs (siRNAs), or siRNAs specific for raptor or rictor, respectively. Total protein lysates were used to prove the downregulation of endogenous raptor and rictor protein through western blot analyses. In addition, the amount of phosphorylated S6 S240/244, which is a substrate of the mTORC1-regulated p70S6 Kinase, total S6 protein, phosphorylated Akt S473, which is a substrate of mTORC2, and total Akt protein has been analysed. Detection of a- tubulin was included to prove equal loading of lysates. (b) The cells described in (a) were induced to form EBs in medium 1 as described in the Materials and methods section. The incidence of EB formation on day 15 was investigated in two independent experiments. Each independent experiment was carried out by seeding 15 hanging drops in triplicate. The incidence is given as percentage (±s.d.) of the number of attached EBs recovered from 15 hanging drops seeded. (c) Representative pictures of CB3-derived EBs on day 15 in medium 1 obtained in the experiments described in (a) and (b). (d) The size of the EBs obtained in the experiments described in (a–c) was determined using the cell’D software from Olympus and is given in mm2 (±s.d.).

process of hAFSCs did not allow conclusions concern- quality rather than in the quantity of hAFSC-derived ing the specific roles of mTORC1 and mTORC2 in this EB development and show that mTORC1 and process. To investigate this issues in more detail, we mTORC2 differently affect this process. The latter is carried out experiments using raptor (mTORC1)-speci- not surprising considering the different biological fic siRNA and rictor (mTORC2)-specific siRNA. functions and targets of the two mTOR-containing Specifically downregulating endogenous raptor protein complexes (see above). Still, it was interesting to see negatively affected the incidence of formation and the that under the experimental conditions used here, size of hAFSC-derived EBs to an extent that was very modulating raptor indeed specifically affected only the comparable with the effects of rapamycin. Treatment mTORC1-regulated S6 phosphorylation, whereas with rictor-specific siRNA also triggered very compar- modulating endogenous rictor had effects on both able effects on EB size, but was without significant the mTORC2-mediated Akt phosphorylation and consequences on the incidence of EB formation. These mTORC1-regulated S6 phosphorylation. The latter data provide evidence that mTORC2 is involved in the could be due to the fact that mTORC2 also functions

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 975 upstream of mTORC1. As described, mTORC2 is 96-well plates. hAFSCs were seeded (2000 cells per well) into responsible for the full activation of Akt. And Akt- nonadhesive-surface 96-well plates (Sterilin, Caerphilly, UK, mediated phosphorylation downregulates the GTPase- 612V96) using medium 1 containing 0.3% methylcellulose. activating potential of tuberin toward Rheb, which is a Aggregation was promoted by centrifugation and EBs were potent regulator of mTORC1 (Wullschleger et al., 2006; collected on day 6 after inducing the aggregation. Guertin and Sabatini, 2007; Yang and Guan, 2007). However, our obtained data strongly suggest that the Reverse transcriptase–PCR mTORC2-mediated effects on the process of EB RNA was prepared using the Cell-to-cDNA-II kit (Ambion, formation cannot exclusively be attributed to its Austin, TX, USA). RNA was checked for DNA contamina- potential to regulate mTORC1, as we observed pro- tion using GAPDH primers specific for DNA recognition and nounced differences between the effects of modulating transcribed into cDNA following the Cell-to-cDNA-II kit mTORC2 activity and modulating mTORC1 on the manufacturer’s instructions. The PCR reactions were prepared incidence of EB formation. to contain 0.2 mM dNTPs and 1.25 U GoTaq-Flexi-DNA polymerase in the according reaction buffer provided by In the last years, evidence has been provided for a role Promega (Promega, Madison, WI, USA). To the reaction, of mTOR in a wide variety of different human diseases 2 mM of each primer was added (Prusa et al., 2003). The used (Dann et al., 2007; Rosner et al., 2008). Our findings primers, published to be specific earlier, are listed below with reported here that they can form EBs and that mTOR the expected product sizes in parentheses (Chunhui et al., 2001; has a major role in this process identify hAFSCs as a Sato et al., 2004; Ng et al., 2005; Takahashi et al., 2007; human stem cell model, which can be used to investigate Ungrin et al., 2008): the role of alterations of the mTOR pathway in studies of the three-dimensional and tissue-level contexts of a variety of differentiation processes, thereby enabling Oct4 (169bp) Forward: 50-CTTGCTGCAGAAGTGGGTGGAG 0 more detailed investigations of mTOR-associated dis- GAA-3 Reverse: 50-CTGCAGTGTGGGTTTCGGGCA-30 eases and drug development. GATA4 Forward: 50-TCCCTCTTCCCTCCTCAAATTC-30 (194bp) Reverse: 50-TCAGCGTGTAAAGGCATCTG-30 Materials and methods Flk1 (400 bp) Forward: 50-CATATCTGTCCTGATGTGATAT GTC-30 Reverse: 50-CATAGCATGTCTTATAGTCATTG Cells, cell culture, flow cytometry and cytogenetics TTC-30 The stem cell lines, CB3 and CD117/2, used here were HBE1 (126 bp) Forward: 50-TGCATGTGGATCCTGAGAAC-30 established by isolation of hAFSCs through magnetic cell Reverse: 50-CGACAGCAGACACCAGCTT-30 sorting using the CD117 MicroBead Kit (Miltenyi Biotec, Pax6 (317 bp) Forward: 50-ACCCATTATCCA- Bergisch Gladbach, Germany, 130–091–332) (De Coppi et al., GATGTGTTTGCCCGAG-30 2007). This project has been reviewed and accepted by the Reverse: 50-ATGGTGAAGCTGGGCATAGGCG ethics commission of the Medical University of Vienna, GCAG-30 T (274 bp) Forward: 50-GCCCTCTCCCTCCCCTCCACGCA Austria (project number: 036/2002). The clonal Q1 hAFSC 0 line has already been established earlier (De Coppi et al., CAG-3 Reverse: 50-CGGCGCCGTTGCTCACAGACCA 2007). IMR-90 fibroblasts were obtained from the American CAGG-30 Type Culture Collection (Rosner and Hengstschla¨ger, 2008). E-cadherin Forward: 50-CCTGGTTCAGATCAAATCCAA Cells were harvested and fixed by rapid submersion in ice-cold (129 bp) C-30 85% ethanol, DNA was stained with 0.25 mg/ml propidium Reverse: 50-GTCACCTTCAGCCATCCTG-30 iodide, 0.05 mg/ml RNase, 0.1% Triton X-100 in citrate buffer, b-Actin (401 bp) Forward: 50-ACAGCAGTCGGTTGGAGCGAGC pH 7.8, and analysed on a Beckton Dickinson FACScan ATC-30 (Becton Dickinson, San Jose, CA, USA) (Rosner et al., 2003). Reverse: 50-CAAGTCAGTGTACAGGTAAGCC CTG-30 Cell numbers and cell size were determined on a hemocyt- 0 0 ometer or on a Casy Cell Counter (Scha¨rfe System, Innovatis, Nodal (126 bp) Forward: 5 -ACCGAGTCCCTTCCACTTGTTG-3 reverse: 50-AGAGGCACCCACATTCTTCCAC-30 Reutlingen, Germany). Chromosome banding was produced by means of a conventional 550-band trypsin–Giemsa analysis (Hengstschla¨ger et al., 2005). Immunocytochemistry Cells or EBs were fixed in 4% paraformaldehyde for 10 min at EB formation 4 1C, treated with 0.5% Tween 20 and blocked for nonspecific Human amniotic fluid stem cells were harvested and resus- binding with phosphate-buffered saline containing 1% bovine pended in either medium 1 or medium 2 (Table 1), and two serum albumin and 0.5% Tween 20 for 30 min at room standard methods were used to induce EB formation: the temperature. Overnight incubation at 4 1C was performed with hanging drop method (Narita et al., 1996; Koike et al., 2007) anti-Oct4 antibody (Chemicon, Billerica, MA, USA, and cultivation in suspension in low-adherence 96-well plate MAB4305), anti-Flk1 antibody (Abcam, Cambridge, UK, (Ng et al., 2005). For the hanging drop method, 20 ml drops ab39256), anti-nestin antibody (Neuromics, Edina, MN, USA, containing 4000 cells in suspension were placed on the lid of a MO15012), anti-laminin antibody (Sigma-Aldrich, St Louis, 100-mm Petri dish. The lid was inverted and placed over the MO, USA, L9393), anti-a-fetoprotein antibody (R&D System, bottom of a Petri dish filled with phosphate-buffered saline. Minneapolis, MN, USA, MAB1369) or anti-E-cadherin anti- On day 4, the EBs formed were transferred on 0.1% gelatin- body (BD biosciences, San Jose, CA, USA, 610181). After coated 100-mm tissue culture dish. Growth media were washing with phosphate-buffered saline, 0.5% Tween 20 cells renewed every 3 days and EBs were kept in culture up to 15 and EBs were incubated with secondary antibodies (anti- days. EBs were also formed in suspension using low-adherence mouse IgG Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA,

Oncogene EB formation of hAFSCs depends on mTOR A Valli et al 976 or anti-rabbit IgG Alexa Fluor 488, Invitrogen) for 4 h at TX, USA, A120-101P); mouse monoclonal antibodies were room temperature. Nuclei were counterstained with 4,6- detected using anti-mouse IgG, an HRP-linked heavy and light diamidino-2-phenylindole (1 mg/ml). chain antibody from goat (A90-116P, Bethyl Laboratories). Signals were detected using the enhanced chemiluminescence Microscopy method (Pierce, Rockland, IL, USA) (Rosner and Hengsts- Immunofluorescence analysis of cells and EBs was carried out chla¨ger, 2008). with a Zeiss LSM5 Exciter confocal microscope (Zeiss, Oberkochen, Germany). The size of the EBs was determined Statistical analyses using the cell’D software from Olympus (Olympus Austria, Flow cytometry data, Casy Cell Counter (Scha¨rfe System, Vienna, Austria). Innovatis, Reutlingen, Germany) results, incidence of EB formation and EB size data are presented as average ±s.d. siRNA treatment Comparisons between groups were carried out by using RNA silencing was achieved using human raptor and rictor Student’s t-test (paired, two-tailed) using Graph-Pad INSTAT siRNA (Dharmacon, Lafayette, CO, USA) at a final concen- software (GraphPad Software Inc, La Jolla, CA, USA) with tration of 50 nM. siRNA was delivered to the cells using P-values o0.05 indicating statistical significance. RNAiMAX reagent (Invitrogen) following the transfection protocol provided by the manufacturers. A pool of four nontargeting siRNAs was used as a control for nonsequence- specific effects. Conflict of interest

Protein extraction and immunoblotting The authors declare no conflict of interest. Protein samples were extracted as described earlier (Rosner et al., 2007) and run on an SDS–polyacrylamide gel and transferred to nitrocellulose. For immunodetection, antibodies specific for the following proteins were used: phospho-S6 Acknowledgements ribosomal protein S240/244 (Cell Signaling, Danvers, MA, USA, #2215), S6 (Cell Signaling, #2317), rictor (Cell Signaling, Research in our laboratory is supported by the FWF Austrian #2114), raptor (Cell Signaling, #2280), phospo-Akt S473 (Cell Science Fund (P18894-B12), by the Herzfelder’sche Familien- Signaling, #4060), Akt (Cell Signaling, #9272) and a-tubulin stiftung and by the Research Training Network ‘Developing (Calbiochem, La Jolla, CA, USA, #CP06). Rabbit polyclonal a stem cell based therapy to replace nephrons lost through and monoclonal antibodies were detected using anti-rabbit reflux nephropathy’ (http://www.kidstem.org) funded by the IgG, a horseradish peroxidase (HRP)-linked heavy and light European Community as part of the Framework program 6 chain antibody from goat (Bethyl Laboratories, Montgomery, (FP6 036097–2).

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