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EMBRYONIC STEM CELLS Insulin Redirects Differentiation from Cardiogenic Mesoderm and Endoderm to Neuroectoderm in Differentiating Human Embryonic Stem Cells CHRISTIAN FREUND,a DORIEN WARD-VAN OOSTWAARD,a,b JANTINE MONSHOUWER-KLOOTS,a STIENEKE VAN DEN BRINK,a MARGA VAN ROOIJEN,a XIUQIN XU,c,d ROBERT ZWEIGERDT,c,d CHRISTINE MUMMERY,a,b ROBERT PASSIERa a

Developmental Biology and Stem Cell Research, Hubrecht Institute, Utrecht, The Netherlands; bInteruniversity Cardiology Institute of The Netherlands, Utrecht, The Netherlands; cES Cell International Pte. Ltd., Singapore, Republic of Singapore; dInstitute of Medical Biology, Singapore, Republic of Singapore Key Words. Human embryonic stem cells • Cardiomyocyte • Insulin • Akt • Pluripotency

ABSTRACT Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/ insulin-like growth factors and their intracellular downstream target protein kinase Akt are known to protect many cell types from apoptosis and to promote proliferation, including hESC-derived cardiomyocytes. Here, we show that in the absence of insulin, a threefold increase in the number of beating areas was observed in hESC/ END-2 coculture. In agreement, the addition of insulin strongly inhibited cardiac differentiation, as evidenced by a significant reduction in beating areas, as well as in ␣-actinin and ␤-myosin heavy chain (␤-MHC)-expressing cells. Real-time reverse transcription-polymerase chain reaction and Western blot analysis showed that insulin

inhibited cardiomyogenesis in the early phase of coculture by suppressing the expression of endoderm (Foxa2, GATA-6), mesoderm (brachyury T), and cardiac mesoderm (Nkx2.5, GATA-4). In contrast to previous reports, insulin was not sufficient to maintain hESC in an undifferentiated state, since expression of the pluripotency markers Oct3/4 and nanog declined independently of the presence of insulin during coculture. Instead, insulin promoted the expression of neuroectodermal markers. Since insulin triggered sustained phosphorylation of Akt in hESC, we analyzed the effect of an Akt inhibitor during coculture. Indeed, the inhibition of Akt or insulin-like growth factor-1 receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 cocultures, insulin does not prevent differentiation but favors the neuroectodermal lineage at the expense of mesendodermal lineages. STEM CELLS 2008;26:724 –733

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Human embryonic stem cells (hESC) hold great potential for regenerative medicine based on cell replacement therapy and for drug and toxicology screens because of their ability to form many, if not all, cell types of the human body. Genetic modification of hESC may in the near future also allow development of specific disease models. A prerequisite for these applications, however, is the ability to control the developmental fate of hESC such that the differentiation of each cell type is efficient and homogeneous populations can be produced as required. Differentiation begins with lineage commitment to extraembryonic or embryonic lineages followed by further choices to form endoderm, ectoderm, or mesoderm. In this study, we addressed the fine-tuning of the earliest stages of commitment and identified a specific function of insulin in this process. Insulin is commonly present in hESC cultures, either as a direct medium supplement or as an active component of fetal

calf serum (FCS) and knockout serum replacement [1, 2]. Insulin and insulin-like growth factors (IGF) belong to a family of growth factors that mediate their effects through structurally similar receptors. Downstream signaling involves activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase Akt [3]. Among the functions of activated Akt are protection of cells from apoptosis and stimulation of cell proliferation. The proliferation of hESC-derived cardiomyocytes was reported as being mediated by this pathway [4]. In addition, the PI3K/Akt axis has been shown to contribute to self-renewal of undifferentiated hESC [2]. We have reported previously that coculture of hESC with visceral endoderm-like (END-2) cells results in efficient differentiation to mesodermally derived cardiomyocytes and endoderm cells, particularly in the absence of serum [1, 5]. More recently, we have also demonstrated that END-2 cell-conditioned medium supports the differentiation of cardiomyocytes from hESC [6].

Correspondence: Robert Passier, Ph.D., Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Telephone: 31-30-2121-939; Fax: 31-30-251-6464; e-mail: [email protected] Received July 31, 2007; accepted for publication December 9, 2007; first published online in STEM CELLS EXPRESS December 20, 2007. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0617

STEM CELLS 2008;26:724 –733 www.StemCells.com

Freund, Ward-van Oostwaard, Monshouwer-Kloots et al. Here, we show that in the absence of insulin, cardiomyocyte differentiation was significantly increased and that adding insulin to hESC-END-2 cocultures had a very strong inhibitory effect on cardiomyocyte differentiation. Similar results were obtained when hESC were allowed to differentiate through embryoid body (EB) formation, an alternative differentiation procedure, which also leads to cardiomyocyte formation. Effects of insulin were early, with the formation of endoderm, nascent mesoderm, and cardiac mesoderm suppressed. Insulin was not sufficient to maintain hESC in an undifferentiated state in coculture but rather caused a shift from endodermal/mesodermal lineages toward the neuroectodermal lineage. We further provide evidence that these effects are partially mediated by Akt and the insulin-like growth factor-1 receptor (IGF-1R).

MATERIALS

AND

METHODS

Derivation of HESC-NL3 and HESC-NL4 hESC lines HESC-NL3 and HESC-NL4 were derived on human foreskin fibroblasts (HF) by S.v.d.B., D.W.-v.O., and M.v.R. from embryos donated following informed consent by couples undergoing in vitro fertilization treatment in The Netherlands. Fertilized oocytes were cultured to blastocysts in Erasmus Medical Centre-In Vitro Fertilization medium (Cambrex, Charles City, IA, http:// www.cambrex.com) supplemented with 10% human plasma (Sanquin, Amsterdam, The Netherlands, http://www.sanquinreagents. com; a gift from Dr. Wouter van Inzen [Erasmus Medical Centre]). After zona pellucida digestion with acid Tyrode’s solution (MediCult, Jyllinge, Denmark, http://www.medicult.com), the inner cell mass was mechanically isolated and cultured on irradiated HF cells (a gift from ES Cell International, Singapore, http://www. escellinternational.com) in gelatin-coated tissue culture dishes. The culture medium consisted of knockout (KO)-Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% KO-serum replacement, 2 mM glutamine, 1⫻ nonessential amino acids, 25 U/ml penicillin, 25 ␮g/ml streptomycin, 100 ␮M ␤-mercaptoethanol (all from Invitrogen, Paisley, U.K., http://www.invitrogen.com), and 8 ng/ml basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, http://www.peprotech.com).

Characterization of Newly Derived hESC Lines At the time of routine passage, colonies were fixed with 2% paraformaldehyde (PFA) for 30 minutes and immunostained for Oct 3/4 (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http:// www.scbt.com) and Tra-1– 60 (Chemicon, Temecula, CA, http:// www.chemicon.com), as described below. Results are shown in supplemental online Figure 1A. For teratoma formation, clumps of approximately 20 – 40 cells with an undifferentiated morphology were injected into testis of 5-week-old SCID CB-17 mice (Charles River Laboratories, Wilmington, MA, http://www.criver.com). After 8 weeks, the resulting tumors were fixed with 4% PFA, embedded in paraffin, and stained with hematoxylin and eosin. Results are shown in supplemental online Figure 1B.

Cell Culture hESC lines HES3-Envy (constitutively expressing green fluorescent protein [GFP], hereafter named HES3) (passages 110 –140) [7] and HES-2 (passages 38 – 65) [8] were cultured on mitomycin C (10 ␮g/ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com)treated mouse embryonic fibroblasts (MEF) in DMEM containing 20% FCS (HyClone, Logan, UT, http://www.hyclone.com), 2 mM L-glutamine, 1 ⫻ nonessential amino acids, 1:100 insulin-transferrin-selenium, 25 U/ml penicillin, 25 ␮g/ml streptomycin, and 100 ␮M ␤-mercaptoethanol (hESC medium) (all from Invitrogen). HES-2 and HES3 were passaged mechanically once a week with dispase (10 mg/ml in hESC medium; Invitrogen). HESC-NL3 (passages 16 –27) and HESC-NL4 (passages 18 – 48) were cultured as described above and passaged mechanically once a week.

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hESC Differentiation by Coculture and EB Formation END-2 cells were cultured in DMEM/Ham’s F-12 medium supplemented with 7.5% FCS (Cambrex), 1 ⫻ GlutaMAX, 1 ⫻ nonessential amino acids, 25 U/ml penicillin, and 25 ␮g/ml streptomycin (all from Invitrogen) in gelatin-coated tissue culture flasks. At 100% confluence, cells were treated with mitomycin C (10 ␮g/ml; SigmaAldrich) and seeded at a density of 175,000 cells per milliliter into gelatin-coated 12-well plates. For coculture, undifferentiated hESC were treated with dispase as described above, broken into pieces, and placed on END-2 cells. Culture medium was hESC medium without serum and without insulin. Insulin (Invitrogen) was added only when indicated at a concentration of 10 mg/l, unless stated otherwise. IGF-1 (1 mg/l; Sigma-Aldrich) was added when indicated. Medium was changed at days 5, 9, and 12 of coculture except for inhibitor studies (described below). For formation of EBs, hESC were treated with 1 mg/ml collagenase IV (Invitrogen) for 4 minutes at 37°C. hESC colonies were broken up with a pipette tip in a grid-like manner, transferred to a 50-ml tube, and separated from feeders by gravity. Formation of beating areas occurred in lowattachment plates (Corning Enterprises, Corning, NY, http://www. corning.com) when cell aggregates were incubated overnight in hESC medium, followed by 12 days in END-2-conditioned medium. Conditioning was performed for 4 days on confluent END-2 cultures in serum-free hESC medium. Signaling pathway inhibitors were from Calbiochem (Darmstadt, Germany, http://www.emdbiosciences.com; Akt inhibitor VIII) or Cell Signaling Technology (Danvers, MA, http://www.cellsignal.com; MEK 1/2 inhibitor U0126 and PI3K inhibitor LY294002). To avoid accumulation of vehicle (dimethyl sulfoxide [DMSO]), medium was changed daily for the duration of inhibitor addition. The IGF-1R inhibitor NVPAEW541 (Novartis Pharma AG, Basel, Switzerland, http://www. novartis.ch) or DMSO was added at days 0 and 2 of coculture. In general, half of a 12-well plate was used for each experimental condition.

Western Blot Analysis Undifferentiated hESC or hESC cocultured with END-2 cells were excised from underlying MEF or END-2 cells, respectively, and lysed in buffer (20 mM Hepes, pH 7.9, 350 mM NaCl, 20% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P40, 1 mM orthovanadate, 0.1 mM dithiothreitol, 25 mM NaF, 1:1,000 protease inhibitor cocktail; Sigma-Aldrich). Proteins were quantified with the Bio-Rad (Hercules, CA, http://www.biorad.com) protein assay. Twenty micrograms of total protein was resolved on a 10% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and blocked with 5% milk or 5% bovine serum albumin (Roche Diagnostics, Basel, Switzerland, http:// www.roche-applied-science.com) in case of phospho-specific antibodies. Incubation with the following primary antibodies was performed overnight at 4°C: Foxa2, 1:250; brachyury T, 1:500; GATA-6, 1:500; Oct, 1:200; Nkx 2.5, 1:500 (Santa Cruz Biotechnology); Sox2, 1:500 (Chemicon); Gapdh, 1:5,000 (Abcam, Cambridge, U.K., http://www.abcam.com); E-cadherin, 1:1,000 (Invitrogen); ␤-tubulin, 1:1,000 (Covance, Princeton, NJ, http://www. covance.com); and phospho-specific antibodies against Akt, extracellular signal-regulated kinase (ERK)1/2, p38, glycogen synthase kinase-3␤; all 1:1,000 (Cell Signaling Technology). Peroxidase-conjugated antibodies (Cell Signaling Technology) were used at 1:1,000 to 1:5,000 for 1 hour at room temperature. Enhanced chemiluminescence (Pierce, Rockford, IL, http://www.piercenet. com) was the chemical substrate.

Fluorescence-Activated Cell Sorting hESC colonies, excised from END-2 cells, were trypsinized as described for cytospin, fixed with 70% cold ethanol, permeabilized for 5 minutes with 0.1% Triton, and incubated with the primary antibody against ␤-myosin heavy chain (␤-MHC) (1:100; Chemicon) or an isotype-matched control antibody (1:10; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) for 30 minutes on ice. The secondary allophycocyanin-coupled antibody (1:400; Molecular Probes, Eugene, OR, http://probes.invitrogen.

Insulin Determines Differentiation Fate of hESC

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Figure 1. ins (10 mg/l) inhibits the formation of human embryonic stem cell (hESC)derived beating areas. (A): Average number of beating areas per well on d12 of coculture of HES3 and END-2 in presence or absence of ins. ins was added at the beginning of coculture and again at d5 and d9 when the medium was changed. (B): Average number of beating areas per well on d12 of coculture of HES3 and END-2. ins was added daily until d12 starting at the beginning of coculture (d0) or on consecutive days. (C): Average number of beating areas on d12 of coculture of different hESC lines. ins was added daily from the beginning (d0) until d5 of coculture. (D): Percentage of beating HES3 EBs after 12 d in END-2-conditioned medium. ins was present from d0 until d5, as indicated. Data in (A–D) represent an average of at least three independent experiments ⫾ SEM. ⴱ, significant at p ⬍ .05. ⴱⴱ, significant at p ⬍ .005. Abbreviations: d, day; HESC, human embryonic stem cell; ins, insulin.

com) was applied for 30 minutes on ice. Samples were analyzed on a FACSCanto instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Cytospin On day 12 of coculture, hESC colonies were excised from the END-2 cells and trypsinized (0.05% trypsin/0.53 mM EDTA; Invitrogen) for 10 minutes into single-cell suspensions. Fifty thousand cells were spun for 4 minutes at 500 rpm onto a glass slide with a Cytospin 2 instrument (Thermo Fisher Scientific, Waltham, MA, http://www.thermo.com) and fixed with 4% PFA for 15 minutes.

Immunostaining of Differentiated Cells Fixed cells were permeabilized for 8 minutes with phosphatebuffered saline (PBS)/0.1% Triton and blocked with PBS/4% goat serum (DakoCytomation) for 1 hour at room temperature. Incubation with a primary antibody (␣-actinin [Sigma-Aldrich], 1:800; neurofilament 160 [NF160] [Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/⬃dshbwww], 1:800; all in PBS/4% goat serum) was performed overnight at 4°C. A Cy3-labeled secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) was added for 1 hour at room temperature. Immunofluorescence was analyzed by sequential scanning with a confocal scanning laser microscope (Leica, Wetzlar, Germany, http://www.leica.com). For quantification, approximately 100 cytospun cells from 10 randomly chosen areas from three or four independent experiments were counted.

Real-Time Reverse Transcription-Polymerase Chain Reaction Colonies were excised from the MEF or END-2 monolayer, and RNA was isolated using Qiagen RNeasy Mini Kits (Hilden, Germany, http://www1.qiagen.com). DNA was removed by DNase treatment (Qiagen), and cDNA synthesis was performed with 1 ␮g of total RNA by use of the Superscript First Strand Synthesis System (Invitrogen). Each polymerase chain reaction (PCR) was performed in triplicate with the iQ SYBR green supermix (Bio-Rad) on an iCycler instrument (Bio-Rad). Primers were designed with Beacon Designer (Bio-Rad), and specificity was verified by a melting curve analysis and agarose gel electrophoresis of the PCR product. Human acidic ribosomal protein and glucuronidase-␤ were used as housekeeping genes for normalization. Primer sequences are given in supplemental online Table 1.

Statistics Data are presented as mean ⫾ SEM. Statistical analyses were calculated using a Mann-Whitney test or Student’s t test for experiments with high numbers (n). Significance was accepted at the level of p ⬍ .05.

RESULTS Insulin Inhibits Cardiac Differentiation hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line, END-2 [5]. After 12 days of coculture, cardiomyocytes can be readily identified as beating areas. Recently, we reported that, in the absence of serum, a marked increase in the number of beating areas was observed in hESC-END-2 cocultures and that this was particularly important early during differentiation [1]. Under serum-free conditions, depletion of insulin further enhanced the number of beating areas (Fig. 1A). In cocultures of HES3 and END-2 cells, repeated addition of insulin at each normal medium change (days 0, 5, and 9; 10 mg/l) resulted in almost threefold fewer beating areas on day 12 compared with insulin-free conditions (Fig. 1A; average number of beating areas/well: with insulin, 2.6 ⫾ 0.3; no insulin, 7.2 ⫾ 0.4). As insulin is depleted by END-2 during prolonged cell culture (X.Q. Xu, R. Graichen, S.Y. Soo, manuscript submitted for publication), we tested whether the inhibitory effect was greater if insulin was added daily. The formation of beating areas was indeed inhibited even more when insulin was added every day (Fig. 1B, day 0). To determine the kinetics of the inhibitory effect and identify which processes might be affected, insulin was again added daily but starting at different time points (days 1–5; 10 mg/l) and compared with the number of beating areas in insulin-free conditions (Fig. 1B). Insulin strongly inhibited the formation of beating areas when added from the beginning of coculture (day 0) and from day 1 onward, and it still caused a partial inhibition when added from day 2 to day 4 (not significant; Fig. 1B). However, addition from day 5 onward no longer had any effect (Fig. 1B), suggesting that insulin inhibits the formation of beating areas only at early stages of differentiation.

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Figure 2. ins inhibits cardiomyocyte differentiation. (A): Immunostaining of HES3 cells at day 12 of coculture with ins present as in Fig. 1C. Differentiated human embryonic stem cell (hESC) areas were dissected from END-2 cells, dissociated, and cytospun on glass slides. HES3 cells expressed GFP under the control of a constitutive promoter (left panels). Cardiomyocytes were detected with an ␣-actinin-specific primary antibody and a Cy3-coupled secondary antibody (middle panels). Scale bar ⫽ 37.5 ␮m. (B): Quantitative fluorescence-activated cell sorting analysis of cardiomyocytes using ␤-MHC. On day 12 of hESC-END-2 coculture, differentiated hESC areas were dissected from END-2 cells, dissociated, and stained with a ␤-MHC-specific primary antibody and an APC-coupled secondary antibody. Percentages are from three independent experiments. Abbreviations: ctrl, control; GFP, green fluorescent protein; ␤-MHC, ␤-myosin heavy chain; GFP, green fluorescent protein; ins, insulin.

Next, we determined the insulin concentration necessary to block cardiomyocyte formation. When added daily between day 0 and day 5, insulin exerted its inhibitory effect at concentrations between 10 and 1 mg/l but not at 0.1 mg/l or less (data not shown). To investigate whether insulin inhibited differentiation more generally in other hESC lines, three independent hESC lines were exposed to insulin daily during coculture (HES-2, HESCNL4, and HESC-NL3; properties of the recently derived lines HESC-NL3 and HESC-NL4 are shown in supplemental online Fig. 1). Adding insulin from day 0 to day 5 strongly inhibited the formation of beating areas in HES-2 and HESC-NL4 cells (Fig. 1C), as well as in HESC-NL3 cells (data not shown). In addition to insulin, we examined the effect of IGF-1 on cardiomyocyte formation. When added daily in the early phase of coculture, IGF-1 (1 mg/l) also inhibited the formation of beating areas (data not shown). Since END-2 cells are also present in hESC cocultures, they could mediate the insulin inhibitory effect. We therefore repeated experiments using EBs with END-2-conditioned medium, since the hESC lines used in our experiments form cardiomyocytes with only low efficiency in the absence of END-2 cells or conditioned medium (data not shown). Addition of insulin from day 0 to day 5 again strongly inhibited the formation of beating EBs in comparison with insulin-free conditions (Fig. 1D). Thus, insulin blocked the formation of beating areas by acting directly on hESC. To quantify the insulin effect on cardiac differentiation, HES3 differentiated areas from day 12 HES3-END-2 cocultures were excised, dissociated, cytospun onto slides, and stained for the cardiac marker ␣-actinin (Fig. 2A). In randomly selected areas, only a small proportion of (GFP-expressing) hESC were www.StemCells.com

positive for ␣-actinin when cultured with insulin (0.25% ⫾ 0.04%). By contrast, in the absence of insulin, the number of ␣-actinin-positive cells (5.3% ⫾ 0.98%) was increased more than 20-fold. The appearance of a striated pattern of ␣-actininpositive cells confirmed that these cells were cardiomyocytes (data not shown). Fluorescence-activated cell sorting (FACS) analysis of cells stained for another cardiomyocyte marker, ␤-MHC, confirmed the inhibitory effect of insulin (Fig. 2B; ␤-MHC-positive cells: with insulin, 0.6% ⫾ 0.2%; no insulin, 6.8% ⫾ 1.4%; percentages given are of all hESC present).

Insulin Inhibits Differentiation Toward Endoderm and Mesoderm Lineages We recently analyzed time-dependent changes in gene expression patterns by microarray in hESC cocultured with END-2 cells and showed that this reflects early embryonic events, associated with formation of the mesoderm and endoderm lineages. Genes of the ectoderm lineages, however, are hardly expressed [9]. Cardiac precursors derive from specific types of mesoderm during development, and endoderm has been shown to play an important role in this induction process [10]. Since early treatment of cocultures with insulin inhibited cardiac differentiation, we analyzed the effect of insulin on mRNA expression of markers for definitive endoderm and mesoderm in differentiated HES3 areas dissected from END-2 monolayers. In the absence of insulin during coculture, upregulation of endodermal markers, such as Foxa2 and GATA-6, and the mesodermal marker brachyury T was observed. Expression was significantly repressed when insulin was added daily from day 0 to day 5 (Fig. 3A). As transformation of the epiblast epithelium into meso-

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Figure 3. ins represses the expression of endodermal and mesodermal markers. (A): Real-time reverse transcription-polymerase chain reaction of marker transcripts associated with the formation of mesoderm (brachy T), epithelial cells (E-cadherin), definitive endoderm (Foxa2 and GATA-6), and cardiac mesoderm (GATA-4, Nkx 2.5) in UD cells (black columns) or during the time course of differentiation. ins was added daily from d0 until d5 (white columns) or was absent (gray columns). Expression levels represent an average of triplicate determinations in three or four independent experiments ⫾ SEM. ⴱ, significant at p ⬍ .05. (B): Western blot analysis of markers as mentioned in (A) in UD or differentiating HES3 cells. Protein levels of Gapdh served as a loading control. Abbreviations: brachy T, brachyury T; d, day; ins, insulin; UD, undifferentiated.

derm involves loss of E-cadherin [11], we tested whether Ecadherin expression was affected by insulin. Although insulin did not significantly alter mRNA levels of E-cadherin (Fig. 3A), E-cadherin protein levels were indeed higher compared with insulin-free conditions (Fig. 3B). Next, we analyzed the expression of the zinc-finger transcription factor GATA-4 and the homeobox-domain transcription factor Nkx2.5, which play important roles in cardiac development and differentiation. In the absence of insulin, GATA-4 expression displayed a biphasic pattern that was significantly inhibited by the presence of insulin (Fig. 3A). Nkx2.5 expression occurred late during differentiation and was completely blocked by insulin (Fig. 3A). Analysis

of protein expression by Western blot confirmed these results (Fig. 3B). This indicated that insulin suppresses the expression of early markers for endoderm, mesoderm, and cardiac mesoderm.

Insulin Does Not Prevent Differentiation but Favors Neuroectodermal Differentiation Since insulin blocked mesodermal and cardiac differentiation, we sought to determine whether addition of insulin to cocultures delayed differentiation and maintained hESC in an undifferentiated state. Undifferentiated hESC cultured on mouse embryonic feeders displayed a flattened, homogeneous morphology,


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Figure 4. ins does not block differentiation in general. (A): Morphology of HES3 after 7 d on mouse embryonic fibroblasts or END-2 cells in the presence or absence of ins. Scale bars ⫽ 1 mm (left panel) and 0.5 mm (right panel). (B): Real-time reverse transcription-polymerase chain reaction analysis of pluripotency markers Oct 3/4 and nanog in UD cells (black columns) and during the time course of differentiation. ins (10 mg/l) was added daily from d0 until d5 (white columns) or was absent (gray columns). Expression levels represent an average of triplicate determinations in three independent experiments ⫾ SEM. ⴱ, significant at p ⬍ .05. (C): Western blot analysis for Oct 3/4 or Sox2 in UD or differentiating HES3 cells. Protein levels of Gapdh served as a loading control. Abbreviations: d, day; ins, insulin; UD, undifferentiated.

with an area of differentiated cells at the center of each colony (Fig. 4A, left panel); the differentiated cells were routinely removed at passaging. During differentiation on END-2 cells in the absence of insulin, hESC developed three-dimensional outgrowths and, occasionally, cystic structures (Fig. 4A, right panel). These morphological features of differentiation were not blocked when insulin was added during coculture (Fig. 4A, middle panel). Next, we examined the effect of insulin on mRNA expression of two transcription factors Oct 3/4 and nanog, which are known to be important for stem cell selfrenewal [12, 13]. Expression of Oct 3/4 mRNA gradually declined during coculture and was unaffected by insulin (Fig. 4B). This was confirmed by Oct 3/4 protein levels, as determined by Western blot (Fig. 4C). The general expression pattern of nanog was similar to that of Oct 3/4 (Fig. 4B) except that insulin delayed downregulation of nanog at day 5, although expression was already at low levels compared with undifferentiated colonies (Fig. 4B). In addition, we analyzed the expression pattern of Sox2, another transcription factor involved in stem cell selfrenewal [12]. In the absence of insulin, a rapid downregulation of Sox2 protein was observed (Fig. 4C). In the presence of insulin, however, Sox2 protein expression was maintained, even at the late stages of coculture. Besides its role in pluripotency in cells at pregastrulation stages of development, Sox2 is also involved in the maintenance of neural precursors and possibly in neuronal differentiation [14]. Thus, we determined whether insulin favored the expression of other neuronal marker genes (Fig. 5). First, we observed a significant upregulation of Pax6 mRNA at days 9 and 12 of insulin-treated cocultures (Fig. 5A). Pax6 has been shown to play a role in neurogenesis [15]. Next, we tested whether markers of mature neurons were also present [16]. mRNA levels of neuron-specific ␤-tubulin at days 9 and 12 tended to be higher in cocultures with insulin present from day 0 until day 5 (Fig. 5B). Protein levels of ␤-tubulin were increased by 3.5-fold at both time points with insulin present (Fig. 5C; quantification not shown). Addition of insulin during the first 5 days of coculture also led to increased mRNA levels of www.StemCells.com

NF160 at day 9 (Fig. 5D; p ⫽ .066). Quantification of cytospun cells staining for NF160 revealed almost threefold more cells when insulin was added from day 0 until day 5 compared with cocultures without insulin (Fig. 5E; NF160-positive cells: with insulin, 2.3% ⫾ 0.6%; no insulin, 0.8% ⫾ 0.2%; percentages given are of all hESC present). Thus, insulin does not delay differentiation in general but enhances expression of neuroectodermal markers.

The Protein Kinase Akt Partially Mediates the Inhibitory Effect of Insulin The two main intracellular pathways mediating insulin action are the mitogen-activated protein kinase (MAPK) pathway, including ERK1/2, and the PI3K/Akt pathway [17]. First, we investigated which of these pathways were also triggered by insulin in hESC. HES3 cocultures from day 1 were treated with insulin for various times, and cell lysates from dissected colonies were subjected to Western blot analysis. Figure 6A shows that MAPKs ERK 1/2 were already phosphorylated under insulin-free conditions (middle panel). Insulin moderately increased ERK1/2 phosphorylation at 15 minutes; this increase was not maintained at later time points (Fig. 6A). To test whether ERK1/2 mediated the early blocking effect on cardiac differentiation, an inhibitor of MEK1/2, a MAPK kinase upstream of ERK1/2, was added simultaneously with insulin during the first 5 days of coculture. The number of beating areas was determined on day 12. Although the inhibitor efficiently blocked ERK1/2 phosphorylation, it was unable to counteract the inhibitory effect of insulin and restore the number of beating areas (data not shown). In contrast to ERK1/2, Akt phosphorylation under insulin-free culture conditions was low (Fig. 6A, 6B, upper panels). Insulin led to a marked increase in phosphorylation of Ser473 of Akt at 15 minutes, which was maintained for at least 3 hours (Fig. 6A) and even up to 24 hours, the last time point examined (data not shown). Phosphorylation of Ser473 has been shown to be essential for Akt activation [18]. To

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Figure 5. ins favors neuroectodermal outcome. (A): Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) analysis of Pax6 in UD cells (black column) and at d9 and d12 of coculture. ins was added daily from d0 until d5 (white columns) or was absent (gray columns). Expression levels represent an average of triplicate determinations in three independent experiments ⫾ SEM. ⴱ, significant at p ⬍ .05. (B): Real-time RT-PCR analysis of ␤-tubulin with conditions as described in (A). (C): Western blot analysis of ␤-tubulin in HES3 cells at d9 and d12. Protein levels of Gapdh served as a loading control. (D): Real-time RT-PCR analysis of NF160 with conditions as described in (A). (E): Immunostaining of NF160 in cytospun HES3 cells at d12 of coculture in the absence or presence of ins (d0 until d5; left panels; scale bar ⫽ 25 ␮m) and quantification of NF160-positive cells (right panel; percentages given of all HES cells). ⴱ, significant at p ⬍ .05. Abbreviations: d, day; ins, insulin; NF160, neurofilament 160; UD, undifferentiated.

investigate whether inhibition of Akt could rescue the blocking effect of insulin, we used two inhibitors, the PI3K inhibitor LY294002 and the Akt inhibitor VIII. LY294002 effectively blocked insulin-induced Akt phosphorylation, but it also led to an increase in phosphorylation of the MAPKs ERK1/2 and p38 (data not shown). As p38 has been shown to play a role in cardiomyogenesis [19, 20], we looked for a more specific inhibitor. The Akt inhibitor VIII inhibited phosphorylation of Akt and its downstream target GSK-3␤ but did not enhance phosphorylation of the MAPKs ERK1/2 or p38 (Fig. 6B). Thus, to analyze the role of Akt in cardiomyogenesis, the Akt inhibitor VIII or vehicle (DMSO) was added simultaneously with insulin during the first 5 days of coculture, and beating areas were counted on day 12. As shown in Figure 6C, inhibition of Akt led to a marked increase in ␤-MHC-positive cells compared with insulin alone, as determined by FACS. This effect was seen only with an inhibitor concentration of 1 ␮M. At higher concentrations (e.g., 2.5 and 10 ␮M), the inhibitor caused adverse effects, such as increased cell death, and no longer rescued the blocking effect of insulin (data not shown). Indeed, Akt is a known survival factor that protects cells from apoptosis [3]. Although we found that a concentration of 1 ␮M was sufficient to block insulin-induced phosphorylation of Ser 473 completely (Fig. 6B), higher concentrations could have nonspecific side effects on other pathways. Besides partially rescuing the inhibitory effect of insulin on the number of ␤-MHC-positive cells, inhi-

bition of Akt also led to an increase in the number of beating areas (supplemental online Fig. 2A). In agreement, we observed that inhibition of Akt partially rescued the expression of Nkx2.5 (Fig. 6D). Furthermore, inhibition of Akt caused a clear downregulation of E-cadherin and Sox2. These results suggested that Akt is an important downstream effector, mediating the insulininduced promotion of neuroectoderm formation and inhibition of cardiac mesoderm.

Inhibition of hESC Cardiomyogenesis Is Mediated by the IGF-1R At the concentrations that we used to block cardiomyocyte formation in the coculture system, insulin could possibly bind to two signal transduction receptors, namely the insulin receptor (IR) and the IGF-1R [21]. To investigate which receptor might mediate the blocking effect of insulin, we used NVP-AEW541, which is a low molecular weight inhibitor of IGF-1R [22]. Indeed, NVP-AEW541 inhibited insulin-induced phosphorylation of Akt in a concentration-dependent manner (supplemental online Fig. 2B). When applied at days 0 and 2 of coculture in parallel with insulin (day 0 until day 5; 10 mg/l) NVP-AEW541 counteracted the effect of insulin at concentrations between 0.1 and 0.25 ␮M, as assessed by FACS analysis of ␤-MHC (Fig. 6E; n ⫽ 2) and quantification of beating areas (supplemental online Fig. 2C; n ⫽ 2). These concentrations are in the range of the 50% inhibitory concentration (IC50) value for the IGF-1R


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Figure 6. The inhibition of Akt partially reverses the effects of ins. (A): Western blot analysis for p-Akt and p-ERK1/2 after ins stimulation in HES3 cells at day 1 of coculture. Protein levels of Gapdh served as a loading control. (B): HES3 cells were preincubated for 2 hours with the Akt inhibitor (1 ␮M) or vehicle (DMSO) and stimulated with ins for 15 min, as indicated. Cell lysates were subjected to Western blot. (C): Quantitative flow cytometry analysis for ␤-MHC. The Akt-inh. or DMSO was added in parallel to ins from the beginning of coculture until day 5, as indicated. Results are from three independent experiments ⫾ SEM. ⴱ, significant at p ⬍ .05. (D): Western blot analysis for E-cadherin, Nkx2.5, and Sox2 in HES3 cell lysates on days 4 and 12 of coculture. The Akt-inh. or DMSO was added in parallel to ins from the beginning of coculture until day 5, as indicated. (E): Inhibition of the insulin-like growth factor-1 receptor (IGF1R) rescued the effect of ins in a concentration-dependent manner. Shown is quantitative flow cytometry analysis for ␤-MHC. The IGF-1R inhibitor NVP or DMSO was added at days 0 and 2 of coculture at the concentrations indicated, in parallel to ins (day 0 until day 5; n ⫽ 2). Abbreviations: Akt-inh., Akt inhibitor; DMSO, dimethyl sulfoxide; ERK, extracellular signal-related kinase; GSK, glycogen synthase kinase; ins, insulin; ␤-MHC, ␤-myosin heavy chain; min, minutes; NVP, NVP-AEW541; p, phosphorylated.

(0.086 ⫾ 0.028 ␮M) but are still far below the IC50 value for the IR (2.3 ⫾ 0.163 ␮M) [22]. Thus, the inhibition of cardiomyocyte formation by insulin is possibly mediated mainly by the IGF-1R.

DISCUSSION Controlling the differentiation fate of hESC is likely to be dependent on learning how to fine-tune signaling pathways in the undifferentiated cells and their immediate early derivatives. We report here that insulin, acting primarily via the IGF-1R and PI3K/Akt, plays a critical role in the early fate decisions. Insulin blocked cardiomyocyte differentiation induced by coculture with visceral endoderm-like cells. Insulin acted directly on hESC and suppressed the expression of endoderm, mesoderm, and markers of cardiac mesoderm while favoring the expression of neuroectodermal markers (Fig. 7). We showed that insulin interfered with cardiomyocyte differentiation at early stages. One potential mechanism may be the maintenance of higher E-cadherin protein levels by insulin. E-cadherin is a cell-adhesion molecule that contributes to cohesive interactions between epithelial cells and prevents their motility [11]. During gastrulation, the first epithelial-to-mesenchymal transition (EMT) occurs with the formation of mesowww.StemCells.com

derm. This involves the downregulation of E-cadherin and the migration of cells through the primitive streak. The increase of E-cadherin by insulin could disturb the normal process of EMT, precluding correct formation of mesoderm, a prerequisite for cardiogenesis. This notion is supported by our finding that insulin decreased expression of the mesodermal marker brachyury T, indicating that it acted before mesoderm commitment had taken place. Our finding that insulin and the PI3K/Akt axis have a negative impact on early events in cardiomyocyte differentiation is not contradictory to a previous report where a role for IGF/ PI3K/Akt axis in hESC-derived cardiomyocyte proliferation was observed [4]. In that study, PI3K/Akt was triggered after differentiation of hESC into cardiomyocytes. This suggests that PI3K/Akt may have a dual role in early heart development. Whereas activation of this signaling pathway seems to be counteractive during the very early steps by preventing the development of mesendoderm, it may be required to stimulate proliferation once immature cardiomyocytes have emerged. This highlights the notion that effective differentiation of hESC into specific cell types requires precise timing of inducing factors. We also showed that insulin strongly enhanced phosphorylation of Akt in hESC during coculture. One of the best-known functions of Akt is to protect cells from apoptosis. For instance, Akt promotes cell survival by phosphorylation of proapoptotic proteins such as BAD, thus inhibiting their binding to prosur-

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Figure 7. Schematic representation of the role of insulin/Akt in determining fate control in early differentiation of human embryonic stem cells. Insulin inhibits END-2-induced cardiomyocyte differentiation by suppressing formation of endoderm and mesoderm, as well as cardiac mesoderm. A potential mechanism could be the maintenance of high E-cadherin levels by insulin, which disturbs normal EMT during gastrulation. Insulin favors the formation of neuroectodermal lineages. Pharmacological inhibition of protein kinase Akt and of the insulin-like growth factor-1 receptor reverses the insulin-dependent effects. Abbreviations: EMT, epithelial-to-mesenchymal transition; IGF-1R, insulinlike growth factor-1 receptor; NVP, NVP-AEW541.

vival proteins [3]. We did not observe an increase in cell death when insulin was depleted from the medium during coculture (not shown). A possible explanation for the apparent absence of apoptosis could be that although the Akt-survival pathway was not active, there were no proapoptotic signals in this culture model. Our finding that insulin blocks cardiomyocyte formation could be a result of a decrease in cell number. We did not observe decreased proliferation (total cell number) during the first 5 days of differentiation in the presence of insulin (data not shown). However, at day 12 of coculture, the total cell number of hESC was 1.5-fold lower in the presence of insulin than in cocultures without insulin. It is not likely that this accounts for the difference in differentiation to cardiomyocytes (20-fold increase in the absence of insulin). This is further corroborated by the fact that the presence of insulin during differentiation from day 5 onward had no inhibitory effect on the formation of beating areas. In multiple types of stem cells, the PI3K/Akt axis has been found to promote self-renewal. For instance, activation of Akt signaling was sufficient to maintain pluripotency in mouse and primate ESC [23]. Human ESC differentiated only when PI3K signaling was suppressed by a pharmacological inhibitor [2]. However, our results suggest that the effects of PI3K/Akt may be more subtle. Although insulin triggered a long-term phosphorylation of Akt, this was not sufficient to maintain pluripotency. First, insulin did not block the development of the typical morphology of differentiated cells. Second, downregulation of the pluripotency markers Oct 3/4 and nanog was not significantly delayed by insulin in comparison with the insulin-free cocultures. However, the expression of Sox2, another transcription factor involved in stem cell self-renewal, was strongly enhanced when insulin was present. To drive the expression of

Insulin Determines Differentiation Fate of hESC pluripotency genes, Sox2 needs cooperative interaction with Oct 3/4 [24]. The reason that insulin was unable to maintain hESC in an undifferentiated state in our model could be the gradual loss of Sox2-interaction partner Oct 3/4. Favoring this hypothesis is the parallel decline of nanog, a known target gene of Sox2/Oct3/4 [25]. Our results suggest that besides its known role in the maintenance of self-renewal, insulin can modulate cellular fate under certain conditions. We used visceral endoderm-like END-2 cells to induce cardiomyocyte differentiation in hESC. Endoderm has been shown to play an important role in the induction of cardiogenesis in chick and Xenopus [26, 27]. Cardiac induction with END-2-conditioned medium suggests that soluble factors rather than cell-cell contacts play a principal role in this process. Under these conditions, the insulin PI3K/Akt axis is no longer sufficient to maintain pluripotency. This highlights the notion that the role of a single pathway depends on the activation status of other molecules in the cellular signaling network. Additional studies are needed to determine crosstalk of signaling pathways controlling pluripotency and differentiation in hESC. Our data indicate that insulin favors differentiation of neuroectodermal lineages. Depending on the nature of its interaction partners, Sox2 involvement has been shown not only in the maintenance of pluripotency of ES cells but also in precursors committed to the neuronal lineage, where it blocks further maturation [28]. Other results suggest that Sox2 could also be involved in neuronal differentiation [14]. In addition, Sox2 plays a role in the development of other ectodermal tissues, such as the eye. In this context, the transcription factor Pax6 is an important interaction partner [29]. The expression of the first lens-specific gene ␦-crystallin depends on the cooperative action of Sox2 and Pax6 [29]. Interestingly, we found partial overlap in the expression of Pax6 and Sox2 in insulin-treated cocultures. Pax6 was upregulated from day 7 onward (data not shown), when Sox2 expression levels were still elevated. Thus, between day 7 and day 9, Sox2 and Pax6 could cooperate in transcriptional regulation. Besides its role in eye formation, Pax6 has been shown to play a key role in the development of the pancreas and nervous system, such as the forebrain in mice, where the absence of Pax6 leads to reduced expression of transcription factors involved in neuronal differentiation [30]. Thus, at later time points, Pax6 alone could regulate the expression of neuronal genes. In the presence of insulin, we also observed significantly more cells expressing markers of mature neurons, although absolute numbers were low. Thus, insulin seems to favor neuroectodermal cell fate, with most cells possibly still in an immature stage. An involvement of insulin receptor-binding molecules in the induction of neuroectodermal structures has been shown before. Three receptor types that bind insulin have been described, namely the IR itself, the IGF-1R, and the IGF-2R [21]. Whereas binding of insulin to IR is important in metabolism, IR does not seem to play an obvious role in neurogenesis, since mice with a neuron-specific disruption of the Ir gene do not exhibit defects in brain development or neuronal survival [31]. By contrast, Igf1r⫺/⫺ mice have reduced brain size and altered brain structures, including an increased density of neurons in the brain stem and spinal cord [21]. Effects of Igf1 and the Igf1r on the central nervous system have been attributed to proliferation, survival, and differentiation [32]. hESC express both the IR and the IGF-1R at a protein level (not shown). In our experiments, we used insulin at a concentration of 10 mg/l, which corresponds to approximately 1.5 ␮M. At this concentration, it is likely that insulin also binds to the IGF-1R. In line with that, we found that a low molecular weight inhibitor of the IGF-1R could rescue the blocking effect of insulin on cardiomyocyte formation. In summary, we show here

Freund, Ward-van Oostwaard, Monshouwer-Kloots et al. that the insulin-Akt pathway plays an important role in determining differentiating cell fate in hESC and that routine addition of insulin in growth factor supplements can inadvertently affect the outcome of differentiation protocols.

ACKNOWLEDGMENTS We thank Dr. Leon Tertoolen for the introduction to confocal microscopy, Laura Zeinstra for expert technical help, and Dr. Wouter van Inzen (In Vitro Fertilization, Erasmus University, The Netherlands) for embryo donation. The antibody against ␤-tubulin was a kind gift of Dr. Oliver Bru¨stle (Bonn, Ger-

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many). We also thank Dr. Carlos Garcia-Echeverria (Novartis Pharma AG) for providing us with the IGF-1R inhibitor NVPAEW541. Financial support was from ES Cell International (to C.F. and J.M.-K.), Dutch Platform for Tissue Engineering (to C.F. and S.v.d.B.), and the European Community’s Sixth Framework Programme contract (“HeartRepair”) LSHM-CT2005-018630 (to R.P.).

DISCLOSURE

OF POTENTIAL OF INTEREST

The authors indicate no potential conflicts of interest.

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