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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM (IPS) CELLS
Mouse Embryonic Stem Cells Established in Physiological-Glucose Media Express the High KM Glut2 Glucose Transporter Expressed by Normal Embryos JIN HYUK JUNG, XIAO DAN WANG, MARY R. LOEKEN Key Words. Embryonic stem cells • Stem cell culture • Cell culture • Glucose transporter
Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA Correspondence: Mary R. Loeken, Ph.D., Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, 1 Joslin Place, Boston, Massachusetts 02215, USA. Telephone: 617-3092525; Fax: 617-309-2650; E-Mail:
[email protected] Received May 7, 2013; accepted for publication August 9, 2013; first published online in SCTM EXPRESS October 28, 2013. ©AlphaMed Press 1066-5099/2013/$20.00/0 http://dx.doi.org/ 10.5966/sctm.2013-0093
ABSTRACT Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early postimplantation embryos. Glut2 is important for survival before embryonic day 10.5. The Glut2 KM (⬃16 mmol/liter) is significantly higher than physiologic glucose concentrations (⬃5.5 mmol/liter), suggesting that Glut2 normally performs some essential function other than glucose transport. Nevertheless, Glut2 efficiently transports glucose when extracellular glucose concentrations are above the Glut2 KM. Media containing 25 mmol/liter glucose are widely used to establish and propagate embryonic stem cells (ESCs). Glut2-mediated glucose uptake by embryos induces oxidative stress and can cause embryo cell death. Here we tested the hypothesis that low-glucose embryonic stem cells (LG-ESCs) isolated in physiological-glucose (5.5 mmol/liter) media express a functional Glut2 glucose transporter. LG-ESCs were compared with conventional D3 ESCs that had been cultured only in high-glucose media. LG-ESCs expressed Glut2 mRNA and protein at much higher levels than D3 ESCs, and 2-deoxyglucose transport by LG-ESCs, but not D3 ESCs, exhibited high Michaelis-Menten kinetics. Glucose at 25 mmol/liter induced oxidative stress in LG-ESCs and inhibited expression of Pax3, an embryo gene that is inhibited by hyperglycemia, in neuronal precursors derived from LG-ESCs. These effects were not observed in D3 ESCs. These findings demonstrate that ESCs isolated in physiological-glucose media retain a functional Glut2 transporter that is expressed by embryos. These cells are better suited to the study of metabolic regulation characteristic of the early embryo and may be advantageous for therapeutic applications. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:929 –934
INTRODUCTION Preimplantation and early postimplantation mouse embryos express the facilitative glucose transporter Glut2 (also known as Slc2A2) [1–3]. Unlike other glucose transporters expressed by embryos and most adult tissues whose KM values are near normal blood glucose concentrations (⬃5.5 mmol/liter) [1, 4 – 6], the KM of Glut2 is 17 mmol/liter. Thus, the rate of glucose transport by Glut2 increases in parallel with increasing blood glucose concentrations [7]. During pathologic conditions, such as diabetic pregnancy, Glut2 mediates enhanced glucose uptake by embryo cells [8]. Increased glucose uptake by pre- and postimplantation embryos causes oxidative stress, which can induce apoptosis [9 –15]. Glut2⫺/⫺ embryos are protected from the adverse effects of hyperglycemia [8]. Embryonic stem cell (ESC) lines are routinely derived and cultured in media containing 25 mmol/liter glucose [16 –18]. We previously showed that using physiological-glucose
media to derive new ESC lines from blastocysts reduced oxidative stress and improved the yield of new lines compared with using highglucose media [19]. Thus, ESCs that successfully adapt to a high-glucose culture environment may have lost Glut2 function, whereas ESCs kept in physiological glucose may be protected from loss of Glut2 function. Fuel metabolism can modulate embryo and ESC self-renewal or differentiation [20 –23]. Metabolism by ESCs in culture differs from that of the corresponding cells of the embryo [24, 25]. Thus, continued expression of Glut2 after adaptation to the culture may be necessary for metabolic regulation of self-renewal and differentiation, and to derive ESCs for therapeutic applications that will ultimately be transplanted into a physiological environment. This is particularly true if the therapeutic application of ESC-derived tissues requires glucose responsiveness (i.e., derivation of pancreatic  cells to treat diabetes). Here we tested whether ESCs that were derived in physiological-glucose media
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High KM Glucose Transporter Expression by ESCs expressed a functional Glut2 glucose transporter, as demonstrated by glucose uptake kinetics and physiological effects of high glucose exposure.
MATERIALS AND METHODS Additional methods and associated references are available as supplemental online data.
Establishment of Low-Glucose ESCs
Figure 1. LG-ESCs express higher steady-state levels of Glut2 mRNA and protein than D3 ESCs. (A): Total RNA was extracted from undifferentiated ESCs after 4 days of culture. Glut2 mRNA was assayed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and was normalized to rRNA. (B): Total RNA was extracted after 2 days of selection of neuronal precursors from embryoid bodies. Real-time RT-PCR was performed as described in (A). (C): Whole-cell lysates were made from undifferentiated ESCs after 4 days of culture. Glut2 protein was assayed by immunoblot, and membranes were stripped and reprobed using antiserum against -actin. (D): Whole-cell lysates were made after 2 days of selection of neuronal precursors. Immunoblots were performed as in (C). All assays were performed using triplicate culture wells. Representative immunoblots of individual culture wells are shown. Abbreviations: ESC, embryonic stem cell; LG-ESC, low-glucose embryonic stem cell.
All animal procedures were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center. Lowglucose embryonic stem cells (LG-ESCs) were derived in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY, http://www.lifetechnologies.com) as described [19] from blastocysts from matings of FVB mice (The Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). Irradiated CF-1 mouse embryonic fibroblasts (GlobalStem, Rockville, MD, http://www.globalstem.com) were used as a feeder layer during establishment of the ESC line. All cultures were incubated in 5% O2:5% CO2 (balance room air).
RESULTS AND DISCUSSION LG-ESCs Express a Functional Glut2 Glucose Transporter LG-ESCs were established from blastocysts in low-glucose (1,000 mg/liter; 5.5 mmol/liter) DMEM. LG-ESCs displayed typical embryonic stem cell morphology characteristics of a pluripotent murine ESC line (supplemental online Figs. 1, 2).
Figure 2. Low-glucose embryonic stem cells (LG-ESCs) exhibit high Michaelis-Menten kinetics of 2-deoxy-D-glucose uptake that is not exhibited by D3 embryonic stem cells (ESCs). D3 ESCs or LG-ESCs were incubated with 0.5–20 mmol/liter 2-deoxy-D-glucose containing the fluorescent 2-deoxy-D-glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (NBDG), as described in the supplemental online data. (A–C): D3 ESCs. (D–F): LG-ESCs. (A, D): Uptake kinetics without competitor showing Vmax and KM. (B, E): Uptake kinetics with 0.4 mol/liter cytochalasin B. (C, F): Uptake kinetics with 4.0 mol/liter cytochalasin B.
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Figure 3. High-glucose media induce oxidative stress in low-glucose embryonic stem cells (LG-ESCs) but not in D3 embryonic stem cells (ESCs). (A, B): D3 ESCs were cultured in high-glucose (25 mmol/liter) media while undifferentiated and while forming embryoid bodies, and then were cultured for 2 days to select for neuronal precursors in low-glucose Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12) (containing 7.7 mmol/liter glucose) or high-glucose DMEM/F12 (containing 17.5 mmol/liter glucose). Antimycin A (1 mol/liter) was added to high-glucose media during selection of neuronal precursors as a control for induction of oxidative stress. The antioxidants GSH-EE and vitamin E were added to high-glucose or antimycin A-containing media as indicated. (C, D): LG-ESCs were cultured in low-glucose (5.5 mmol/liter) media while undifferentiated and while forming embryoid bodies, and then were cultured for 2 days to select for neuronal precursors in low-glucose or high-glucose DMEM/F12 as above. Antimycin A (1 mol/liter) was added to low-glucose media during selection of neuronal precursors as a control for induction of oxidative stress. GSH-EE or vitamin E was added to low-glucose or antimycin A-containing media as indicated. Markers of oxidative stress were assayed as described in the supplemental online data. (A, C): Malondialdehyde. (B, D): Reduced glutathione. Data were analyzed by analysis of variance followed by the Newman-Keuls post test, comparing results from low-glucose and high-glucose ⫾ antioxidant-cultured samples (D3 ESCs and LG-ESCs), high-glucose and high-glucose ⫹ AA ⫾ antioxidant-cultured samples (D3 ESCs), or low-glucose and low-glucose ⫹ AA ⫾ antioxidant-cultured samples (LG-ESCs). Significant differences are indicated in each panel. Abbreviations: AA, antimycin A; GSH, reduced glutathione; GSH-EE, glutathione ethyl ester; MDA, malondialdehyde.
We hypothesized that the Glut2 glucose transporter expressed by blastocysts is expressed by LG-ESCs. They were compared with D3 ESCs, which are typical of long-established ESC lines that were isolated and propagated in conventional media containing 4,500 mg/liter (25 mmol/liter) glucose [26 –31]. Glut2 mRNA and protein were assayed in undifferentiated D3 and LGESCs and in neuronal precursors derived from both lines [31]. Neuronal precursors were used as a model of embryonic neuroepithelium that responds to Glut2-mediated glucose transport [8, 11, 32]. In both undifferentiated and differentiating ESCs, Glut2 mRNA was expressed at much higher levels in LG-ESCs than in D3 ESCs (Fig. 1A, 1B), and Glut2 protein was readily detectable in LG-ESCs but not in D3 ESCs (Fig. 1C, 1D). The increased expression of Glut2 mRNA and protein was also observed in three ad-
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ditional LG-ESC lines, both when grown as undifferentiated ESCs and when induced to form neuronal precursors (supplemental online Fig. 3). We next measured the Michaelis-Menten kinetics of 2-deoxy-D-glucose transport by D3 and LG-ESCs. The KM of 2-deoxyD-glucose uptake by D3 ESCs was 4.2 mmol/liter (Fig. 2A). The effects of cytochalasin B, which at 0.4 mol/liter inhibits low KM glucose transport and at 4.0 mol/liter inhibits both low and high KM glucose transport [33, 34], on rates of 2-deoxy-D-glucose uptake were tested. 2-Deoxy-D-glucose uptake by D3 ESCs was inhibited by 0.4 and by 4.0 mol/liter cytochalasin B (Fig. 2B, 2C), indicating that only low KM glucose transport was operational in D3 ESCs. In contrast, the KM of 2-deoxy-2-D-glucose uptake by LG-ESCs was 15.8 mmol/liter (Fig. 2D), and transport was
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High KM Glucose Transporter Expression by ESCs
Figure 4. High-glucose media inhibit Pax3 expression by low-glucose embryonic stem cells (LG-ESCs) but not by D3 embryonic stem cells (ESCs). (A): D3 ESCs were cultured in high-glucose media while undifferentiated and while forming embryoid bodies, and then were cultured to select for neuronal precursors in low- or high-glucose Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12), with or without antimycin A, GSH-EE, or vitamin E, as in Figure 3. Undifferentiated and differentiating neuronal precursor cultures were terminated after 4 days or 2 days, respectively, and then were assayed for Pax3 mRNA by real-time reverse transcription-polymerase chain reaction. (B): LG-ESCs were cultured in low-glucose media while undifferentiated and while forming embryoid bodies, and then were cultured to select for neuronal precursors in low- or high-glucose DMEM/F12, with or without antimycin A, GSH-EE, or vitamin E, as in Figure 3. UD or D cultures were terminated and assayed for Pax3 mRNA as in (A). (C, D): UD and D D3 ESCs (C) and LG-ESCs (D) cultured as in (A) and (B) and assayed for Nestin mRNA. Gene expression by differentiating ESCs was analyzed as in Figure 3. Significant differences are indicated in each panel. Abbreviations: AA, antimycin A; D, differentiating neuronal precursor; GSH-EE, glutathione ethyl ester; UD, undifferentiated neuronal precursor.
inhibited only by 4.0 mol/liter cytochalasin B (Fig. 2E, 2F). These results demonstrate that LG-ESCs express a functional high KM glucose transporter.
High-Glucose-Induced Physiological Effects in LG-ESCs In vivo, hyperglycemia (⬎14 mmol/liter), caused by maternal diabetes, induces oxidative stress in embryos [9, 10]. Hyperglycemia-induced oxidative stress inhibits expression of Pax3 in neuroepithelium, which causes neural tube malformation [10, 11, 32]. Glut2 is necessary for hyperglycemia-induced malformations [8]. ESC-derived neuronal precursors display a gene expression pattern similar to that of neuroepithelium, including expression of Pax3 [30, 32, 35–37]. Previous studies have shown that oxidative stress induced by antimycin A, which stimulates mitochondrial superoxide production [38, 39], inhibits Pax3 expression by D3 ESC-derived neuronal precursors [32]. The effects of low-glucose (7.7 mmol/liter) and high-glucose (17.5 mmol/liter) media on LG-ESCs were studied to determine whether glucose transported by Glut2 is metabolized and stimulates physiological effects that occur in embryos.
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To investigate whether high glucose induces oxidative stress, malondialdehyde, a marker of lipid peroxidation, and reduced glutathione (GSH) were assayed. As shown in Figure 3A and 3B, high glucose did not induce oxidative stress by D3 ESCs. Only antimycin A induced oxidative stress, and the effects of antimycin A were suppressed by the antioxidants GSH and vitamin E. In contrast, high glucose, as well as antimycin A, induced oxidative stress by LG-ESCs, and the effects of both high glucose and antimycin A were suppressed by antioxidants (Fig. 3C, 3D). To investigate whether high-glucose-induced oxidative stress inhibits Pax3 expression, Pax3 mRNA from D3 ESCs and LG-ESCs, before differentiation and after induction of neuronal precursors in low- or high-glucose media, was assayed by reverse transcription-polymerase chain reaction. High glucose did not inhibit Pax3 expression by D3 ESC-derived neuronal precursors, but Pax3 expression was inhibited by antimycin A (Fig. 4A). In contrast, high glucose, as well as antimycin A, inhibited Pax3 expression by LG-ESCs, and these effects were suppressed by antioxidants (Fig. 4B). The effects of high glucose and oxidative stress on Pax3 expression were not due to overall inhibition of STEM CELLS TRANSLATIONAL MEDICINE
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differentiation because there were no effects of high glucose or antimycin A on the neuronal precursor, Nestin [31], by D3 or LG-ESCs (Fig. 4C, 4D). These results indicate that LG-ESCs are responsive to Glut2-mediated transport and metabolism of glucose at high concentrations, as are embryo cells at corresponding stages of development.
Potential Glut2 Function at Normal Glucose Concentrations Although adult tissues that express Glut2, such as pancreatic  cells and liver, transport glucose from high extracellular concentrations [7, 40, 41], the normal glucose concentration surrounding the embryo (⬃5.5 mmol/liter) is much lower than the Glut2 KM. This suggests that the low KM glucose transporters expressed by embryos are responsible for glucose uptake during normal (nondiabetic) circumstances. Nevertheless, Glut2⫹/⫺ and Glut2⫺/⫺ embryos were recovered from euglycemic pregnancies on embryonic day 10.5 at lower than Mendelian frequencies [8, 41]. This suggests that Glut2 is important for early embryo survival for a function other than glucose transport. Glut2 is also a high-affinity glucosamine transporter (KM 0.8 mmol/liter) [42]. Glucosamine is a substrate for protein modification by O-linked-N-acetylglucosamine (O-GlcNAcylation). O-GlcNAcylation of Oct4, Sox2, and phosphofructokinase 1 affects ESC proliferation and anabolic reactions [43, 44]. Glucosamine can be endogenously synthesized from glycolytic intermediates or it can be taken up from circulation [45– 47]. It is possible that embryos express Glut2 for uptake of maternally produced glucosamine to spare glycolytic intermediates for other metabolic needs.
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CONCLUSION An ESC line derived in physiological-glucose media expressed a functional Glut2 transporter that is expressed by normal embryos, whereas an established ESC line that had been isolated and propagated in high-glucose media did not. Glut2-expressing ESCs should be advantageous for the study of metabolic regulation of embryonic development, as well as for transplantation for therapeutic applications.
ACKNOWLEDGMENTS Research reported in this publication was supported by NIH Grant R01-DK052865 to M.R.L. and was assisted by core facilities supported by Diabetes Endocrine Research Center Grant P30DK036836 to the Joslin Diabetes Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
AUTHOR CONTRIBUTIONS J.H.J. and X.D.W.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.R.L.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.
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STEM CELLS TRANSLATIONAL MEDICINE
Mouse Embryonic Stem Cells Established in Physiological Glucose Media Express the High KΜ Glut2 Glucose Transporter Expressed by Normal Embryos Jin Hyuk Jung, Xiao Dan Wang, and Mary R. Loeken
SUPPLEMENTARY INFORMATION
SUPPLEMENTAL MATERIALS AND METHODS ESC Culture and Differentiation LG-ESC were cultured on irradiated MEFs until 5 passages, when they were transitioned to grow in culture dishes coated with 0.1% gelatin (Sigma, St. Louis, MO, http://www.sigmaaldrich.com).
Mouse
D3
ESC
(ATCC,
Manassas,
VA,
http://www.ATCC.org) were cultured in high glucose DMEM (Life Technologies) and incubated 5% CO2 (balance room air) as described [1]. Formation of embryoid bodies and neuronal precursors from D3 and LG-ESC was as described [21, 22] except using low glucose DMEM for LG-ESC. Where indicated, D3 or LG-ESC were cultured with 1 µmol/L antimycin A, 50 µmol/L DL-α-tocopherol acetate, or 1 mmol/L glutathione ethyl ester (GSH-EE) (all from Sigma) during days 1-3 of selection of neuronal precursors from embryoid bodies.
RT-PCR Undifferentiated ESC were harvested after four days of culture, and embryoid bodies were harvested after four days of additional culture using bacterial dishes in low glucose media but without LIF. Differentiating ESC were harvested two days after selecting
1
neuronal precursors from embryoid bodies as described [1, 2]. Total RNA was extracted and reverse transcribed from triplicate culture wells as described [3]. Real-time PCR was performed using primers and probes for Pax3 cDNA [4] or for Glut2, Oct4, Nanog, Sox2, Sox1, smooth muscle actin (α-SMA), α-fetoprotein (AFP), and rRNA (obtained from Life Technolgies). mRNA was expressed relative to rRNA as described [4].
Teratoma Formation 106 LG-ESC were injected into the dorsal flank of SCID mice (The Jackson Laboratory) as described [5]. Tumors were dissected after 4 weeks, or after attaining 20 mm diameter, fixed in Sorensen’s PBS containing 4% formaldehyde, and embedded in paraffin. 5 µm sections were stained with hematoxylin and eosin.
Glut2 Immunoblot Assay Fifty µg of whole cell extracts were analyzed by immunoblot as described [6], using rabbit anti-Glut2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) and mouse anti-β-actin (1:5000, Sigma) as primary antibodies and donkey anti-rabbit IgG (1:5000, GE Healthcare Biosciences, Piscataway, NJ, http://www.gelifesciences.com) and goat anti-mouse IgG (1:3000, Santa Cruz Biotechnology) as secondary antibodies. Horseradish peroxidase (HRP)-coupled secondary antibodies were detected by Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, http://www.perkinelmer.com) and exposed to x-ray film.
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Glucose Transport Kinetics Uptake of 2-deoxy-D-glucose was measured as described [7] using the fluorescent 2deoxyglucose analog, 2-deoxy-2-[(7- nitro- 2, 1, 3- benzoxadiazol- 4- yl)amino]-Dglucose (2-NBDG) (Cayman Chemical, Ann Arbor, MI, https://www.caymanchem.com) as the trace. Briefly, 1.5x104 D3 or LG-ESC were cultured overnight in a 96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplate (Corning, Tewksbury, MA, http://www.corning.com). Cultures were rinsed with KRH buffer then were incubated with 100 µl 0.5-20 mmol/l 2-deoxy-D-glucose containing 0.0125-0.5 mmol/l 2-NBDG in KRH buffer for 10 min. Controls were incubated with 0.5-20 mmol/l unlabeled 2-deoxyD-glucose, or 0.4 or 4 µM Cytochalasin B (Sigma). Reactions were terminated by rinsing three times with PBS. 2-NBDG fluorescence was measured at 485/535 nm (excitation/emission). Cells were solubilized in lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% NP40 and 2 mM EDTA) and protein content was measured using Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, http://www.bio-rad.com).
Oxidative Stress Markers Malondialdehyde (MDA) was assayed spectrophotometrically as described [8] using MDA (Cayman Chemical) as a standard. Reduced glutathione (GSH) was determined from the difference between total glutathione and oxidized glutathione (GSSG) as described [9] using a kit obtained from Cayman Chemical. Markers were expressed relative to protein content using Bio-Rad Protein Assay Dye Reagent as described above.
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Statistical Analysis Prism software v.4 (GraphPad Software, San Diego, CA, http://www.graphpad.com) was used for all analyses. The KΜ and Vmax of 2-deoxy-D-glucose uptake were determined using non-linear curve fitting analysis with the Michaelis-Menten equation. All other data were analyzed from three separate culture dishes by analysis of variance (ANOVA) followed by Newman-Keuls post test. Data are expressed as mean ± SEM.
SUPPLEMENT REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Wu Y, Viana M, Thirumangalathu S, et al. AMP-activated protein kinase mediates effects of oxidative stress on embryo gene expression in a mouse model of diabetic embryopathy. Diabetologia 2012;55:245-254. Lee SH, Lumelsky N, Studer L, et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675-679. Phelan SA, Ito M, Loeken MR. Neural tube defects in embryos of diabetic mice: Role of the Pax-3 gene and apoptosis. Diabetes 1997;46:1189-1197. Chang TI, Horal M, Jain SK, et al. Oxidant regulation of gene expression and neural tube development: Insights gained from diabetic pregnancy on molecular causes of neural tube defects. Diabetologia 2003;46:538-545. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663676. Musi N, Fujii N, Hirshman MF, et al. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 2001;50:921-927. Cheatham B, Vlahos CJ, Cheatham L, et al. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 1994;14:4902-4911. Draper HH, Hardley M. Malondialdehype determination as an index of lipid peroxidation. In: Packer L, Glazer AN, eds. Methods in Enzymology. New York: New York Academic Press; 1990:421-431. Li R, Chase M, Jung SK, et al. Hypoxic stress in diabetic pregnancy contributes to impaired embryo gene expression and defective development by inducing oxidative stress. Am J Physiol Endocrinol Metab 2005;289:E591-599.
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SUPPLEMENTAL FIGURE LEGENDS Figure S1. LG-ESC exhibit stem cell characteristics. A. LG-ESC colonies display typical ESC morphology. Scale bar = 10 µm. B. Real time RT-PCR of stem cell markers, Oct4, Nanog, and Sox2, and the differentiation markers, Sox1 (ectoderm), smooth muscle actin (α-SMA) (mesoderm), and α-fetoprotein (AFP) (endoderm), and expressed relative to rRNA, using RNA from undifferentiated LG-ESC (UD) or embryoid bodies (EB).
Figure S2. LG-ESC form teratomas containing all three germ layers. Hematoxylin and eosin stained sections of teratomas tumors generated in SCID mice from LG-ESC showing tissues derived from ectoderm, mesoderm, and endoderm.
Figure S3. Higher steady state levels of Glut2 mRNA and protein are expressed by four independently isolated LG-ESC lines compared to D3 ESC. A. Total RNA was extracted from undifferentiated ESC (D3 or four independent LG-ESC lines, LG-ESC-1, LG-ESC2, LG-ESC-3, and LG-ESC-4) after four days of culture. Glut2 mRNA was assayed by real-time RT-PCR and was normalized to rRNA. B. Total RNA was extracted after two days of selection of neuronal precursors from embryoid bodies. Real-time RT-PCR was performed as described in A. C. Whole cell lysates were made from undifferentiated ESC after four days of culture. Glut2 protein was assayed by immunoblot and membranes were stripped and re-probed using antiserum against β-actin. D. Whole cell lysates were made after two days of selection of neuronal precursors. Immunoblots were performed as in C. All assays were performed using triplicate culture wells.
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Representative immunoblots of individual culture wells are shown. Please note that the cell line designated LG-ESC-1 in this figure is the one that was studied (designated LGESC) in the main manuscript.
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