Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes
© Zaragosi et al; licensee BioMed Central Ltd. 2008
Received: 25 October 2007
Accepted: 13 February 2008
Published: 13 February 2008
Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.
Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.
These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.
Obesity, which is characterized by an excess of adipose mass, is a major public health-problem. Hypertrophy, i.e. increase in the adipocyte size and hyperplasia, i.e. increase in the adipocyte numbers, are observed in severe obesity. It is now well established that multipotent stem cells exist within adipose tissue throughout the life [1–3] and that an excessive recruitment of these adipose precursor cells could lead to hyperplasia. As opposed to hypertrophy, hypoplasia of adipose tissue is observed in lipodystrophy and is associated with diabetes and hyperlipidaemia. Adipocytes and osteoblasts share the same mesenchymal precursor cell . Adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis where adipocytes develop at the expense of osteoblasts . Therefore, pharmacological molecules that control the pool of adipose stem cells are of great interest.
Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase existing in two isoforms GSK3α and GSK3β, is a key regulator of numerous signalling pathways. In particular, GSK3 has been involved in multiple cellular processes including Wnt and Hedgehog (Hh) pathways. In the activation of the canonical Wnt pathway, inhibition of GSK3 results in dephosphorylation of β-catenin leading to its nuclear accumulation. Inhibition of GSK3 also contributes to activation of the Hh pathway by stabilisation of Gli 2/3 transcription factors, favouring their nuclear translocation and leading to transcription of target genes. Gli1 is one of them and induction of Gli1 gene expression has been characterized as a reliable marker of Hh signalling activity . The role of GSK3 in the differentiation of preadipose cells has been previously described. It has been shown that activation of the Wnt pathway via inhibition of GSK3 inhibits adipogenesis of murine preadipocytes and in mice [7, 8]. Expression of Hh target genes was reduced in fat depots of obese mice, suggesting anti-adipogenic properties of this pathway . GSK3 is also a key component of the circadian apparatus. The circadian clock may play a role in adipocyte metabolism and it has been recently shown that inhibition of GSK3 in human adipocytes lengthened the period of expression of core circadian transcriptional apparatus . Therefore, GSK3 could represent a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. To address this point we have studied the effects of two pharmacological inhibitors of GSK3, lithium chloride (LiCl)  and 6-bromoindirubin-3'-oxime (BIO) , on multipotent stem cells derived form human adipose tissue (hMADS cells, also named ASC as suggested by IFATS, a society focusing on Adipose-derived Stem or Stromal Cells, and discussed by Mitchell et al. . We have previously established the procedure to isolate and expand hMADS cells from different donors. hMADS cells exhibit key features of mesenchymal stem cells such as self-renewal capacity and ability to undergo differentiation at the single cell level into at least two lineages (adipogenic and osteogenic) [14, 15]. Thus, hMADS cells represent a potent cellular model to investigate pathways regulating self-renewal, adipogenesis and osteogenesis [16, 17].
Results and Discussion
Functional inhibition of GSK3 in human adipose-derived stem cells
Effect of GSK3 inhibitors on proliferation of human adipose-derived stem cells
Effects of BIO on differentiation of human adipose-derived stem cells
To investigate the effect of GSK3 inhibition on adipocyte differentiation, hMADS cells were induced to differentiate in the presence of BIO or MeBIO. Adipocytes and osteoblasts share the same mesenchymal precursor cell. Adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis in which adipocytes develop at the expense of osteoblasts [5, 20]. We took advantage of hMADS cells that were previously demonstrated to differentiate in vitro into functional adipocytes and osteoblasts [16, 17], to investigate the effect of GSK3 inhibition on both lineages.
It has been observed that patients treated with LiCl for bipolar disorder display weight gain and reduced risk of fractures, suggesting that LiCl promotes both adipogenesis and osteogenesis in humans in vivo [23, 24]. However, a central effect of LiCl and an indirect effect on adipocytes and osteoblasts in these patients cannot be excluded.
Differential effects of BIO treatment during early steps of adipogenesis and osteogenesis
Identification of mechanisms leading to the differential effects on adipogenesis and osteogenesis of hMADS cells could lead to a preferential use of the GSK3 inhibitors on adipocyte differentiation in vivo. In conclusion, our data strongly suggest that GSK3 is a promising pharmacological target to regulate both the number and differentiation of adipocyte precursors in human adipose tissue. However, we have to keep in mind that potential adverse effects could be observed due to the fact that GSK3 is implicated in numerous signalling pathways. Therefore, it would be important in the future to identify the signalling pathway mediating the adipogenic effect of GSK3.
We have shown that BIO and LiCl, two inhibitors of GSK3, inhibited proliferation as well as adipogenic and osteogenic differentiations of stem cells isolated from human adipose tissue. Data demonstrate the feasibility of a pharmacological approach to regulate adipose-derived stem cell function.
Isolation and culture of hMADS cells
hMADS cells were obtained from the stroma of human adipose tissue as described previously . Adipose tissue was collected, with the informed consent of the parents, as surgical scraps from surgical specimen of various surgeries, as approved by the Centre Hospitalier Universitaire de Nice Review Board. The cell populations that have been studied in this work were isolated from the pubic region fat pad of a 5-year old (hMADS2) and of a 4-month old (hMADS3) male donor. Proliferation medium for routine maintenance of hMADS cells is composed of DMEM (low glucose) containing 10% foetal calf serum, 10 mM HEPES, 100 U/ml penicillin and streptomycin and supplemented with FGF2 as previously reported . After reaching 80% confluence, adherent cells were dissociated in 0.25% trypsin EDTA and seeded at 4500 cells/cm2. Cells have been maintained in low serum concentration for studying effects of GSK3 inhibitors on proliferation. This medium is composed of 60% DMEM low glucose, 40% MCDB-201, insulin (10 μg/ml), transferrine(5 μg/ml), selenium(50 ng/ml), dexamethasone (10-9M), ascorbic sodium acid (50 μg/ml), 2.5 ng/ml FGF2 and supplemented with 0.5% FCS. This medium containing a low serum concentration allows the maintenance of stem cell features of hMADS2 and hMADS3 cells (not shown). Human mesenchymal stromal cells isolated from bone marrow, termed hBMSC, were purchased from Cambrex and used as recommended by the manufacturer. Cultures were maintained at 37°C in a humidified gassed incubator, 5% CO2 in air.
hMADS cell differentiation
Adipocyte differentiation was performed as described previously . Basically, confluent cells were cultured in DMEM/Ham's F12 media supplemented with transferrin (10 μg/ml), insulin (0.86 μM), triiodothyronine (0.2 nM), dexamethasone (1 μM), isobutyl-methylxanthine (100 μM) and rosiglitazone (500 nM). Three days later, the medium was changed (dexamethasone and isobutyl-methylxanthine were omitted). Neutral lipid accumulation was assessed by Oil red O staining . For osteoblasts, confluent cells were cultured in α-MEM medium containing 10% FCS, L-ascorbic acid phosphate (50 μg/ml), β-glycerophosphate (10 mM) and 100 nM dexamethasone. Alizarin red staining was performed as previously described . For simultaneous adipocyte and osteoblast differentiations, confluent cells were cultured in the differentiation medium consisting in 50% adipogenic and 50% osteogenic media.
Measurement of adipogenic and osteogenic specific enzymatic activities
Cell homogenates for both Glycerol-3-phosphate dehydrogenase (GPDH) and Alkaline phosphatase (ALP) enzymatic activity measurements were prepared in 20 mM Tris-HCl pH 7.5 buffer containing 1 mM EDTA and 1 mM 2-mercaptoethanol. GPDH activity was measured by the absorbance at 340 nm as described previously  and ALP activity was assayed using the p-Nitrophenyl Phosphate Liquid Substrate System (Sigma). Absorbance was measured at 412 nm.
Cell proliferation assays
Cells were plated onto 12-well plates (104 cells per well). GSK3 inhibitors were added 24 hours after cell plating in order to avoid a potential effect of the compounds on the efficiency in cell attachment. After the appropriate time, cells were trypsinized as mentioned above and counted with a Coulter counter. For each experiment, three wells per condition were counted.
Cells were plated at a density of 10 cells/cm2 in 100-mm2 dishes. 15 days after plating cells were fixed with 0.25% glutaraldehyde and stained with 0.1% crystal violet. Colonies containing at least 40 cells were enumerated under a light microscope. Medium was changed 3 times a week.
hMADS cells were rinsed twice with PBS at 4°C, and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Free aldehydes were quenched by a 15-min incubation with 1 M glycine in PBS. Fixed cells were permeabilized with 0.5% Triton for 10 min, and unspecific reactions blocked with 5% bovine serum albumin in PBS for 30 min. Cells were then incubated for 1 h with an anti β-catenin (Santa Cruz Biotechnology) antibody diluted in 5% bovine serum albumin in PBS, followed by a 488-Alexa Fluor conjugated secondary antibody. Nuclei were counterstained with DAPI. Images were taken on an LSM510 META confocal microscope (Zeiss).
Total RNA was extracted using TRI-Reagent™ kit (Euromedex, France) according to the manufacturer's instructions and RT-PCR analysis was conducted as described previously . All primers sequences, designed using Primer Express software (Applied Biosystems, France), see Additional file 4. For quantitative PCR, final reaction volume was 25 μl, including specific primers (0.4 μM), 5 ng of reverse transcribed RNA and 12.5 μl SYBR green master mix (Applied Biosystems, France). Quantitative PCR conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 1 min at 60°C. Real-time PCR assays were run on an ABI Prism 7000 real-time PCR machine (Applied Biosystems, France). Normalization was performed using the geometrical average of the housekeeping genes G6PDH, POLR2A and TBP. Quantification was performed using the comparative-ΔCt method. Level of expression was represented using the genesis software . This software generates expression images using a colour code according to the expression intensities.
Statistical significance was checked by using t-tests whenever comparing two conditions or a one-way ANOVA followed by t-tests whenever comparing more than two conditions. Asterisks indicate the significance levels as mentioned in the figure legends.
Authors are grateful to Pr. Gérard Ailhaud for critical reading of the manuscript. This work was supported in part by CNRS, ARC (grant 3721) and the Fondation pour la Recherche Médicale (FRM) funds. The team is "Equipe FRM". LEZ is supported by a fellowship from ARC.
- Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001, 7 (2): 211-228. 10.1089/107632701300062859.View ArticlePubMed
- Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C: The human adipose tissue is a source of multipotent stem cells. Biochimie. 2005, 87 (1): 125-128. 10.1016/j.biochi.2004.11.007.View ArticlePubMed
- Casteilla L, Dani C: Adipose tissue-derived cells: from physiology to regenerative medicine. Diabetes Metab. 2006, 32 (5 Pt 1): 393-401. 10.1016/S1262-3636(07)70297-5.View ArticlePubMed
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284 (5411): 143-147. 10.1126/science.284.5411.143.View ArticlePubMed
- Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M: Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis. Biogerontology. 2001, 2 (3): 165-171. 10.1023/A:1011513223894.View ArticlePubMed
- Cohen P, Frame S: The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001, 2 (10): 769-776. 10.1038/35096075.View ArticlePubMed
- Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA: Inhibition of adipogenesis by Wnt signaling. Science. 2000, 289 (5481): 950-953. 10.1126/science.289.5481.950.View ArticlePubMed
- Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lucas PC, Opp MR, MacDougald OA: Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem. 2004, 279 (34): 35503-35509. 10.1074/jbc.M402937200.View ArticlePubMed
- Suh JM, Gao X, McKay J, McKay R, Salo Z, Graff JM: Hedgehog signaling plays a conserved role in inhibiting fat formation. Cell Metab. 2006, 3 (1): 25-34. 10.1016/j.cmet.2005.11.012.View ArticlePubMed
- Wu X, Zvonic S, Floyd ZE, Kilroy G, Goh BC, Hernandez TL, Eckel RH, Mynatt RL, Gimble JM: Induction of Circadian Gene Expression in Human Subcutaneous Adipose-derived Stem Cells. Obesity (Silver Spring). 2007, 15 (11): 2560-2570.View Article
- Ryves WJ, Harwood AJ: Lithium inhibits glycogen synthase kinase-3 by competition for magnesium. Biochem Biophys Res Commun. 2001, 280 (3): 720-725. 10.1006/bbrc.2000.4169.View ArticlePubMed
- Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, Ryan XP, Vonica CA, Brivanlou A, Dajani R, Crovace C, Tarricone C, Musacchio A, Roe SM, Pearl L, Greengard P: GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol. 2003, 10 (12): 1255-1266. 10.1016/j.chembiol.2003.11.010.View ArticlePubMed
- Mitchell JB, McIntosh K, Zvonic S, Garrett S, Floyd ZE, Kloster A, Di Halvorsen Y, Storms RW, Goh B, Kilroy G, Wu X, Gimble JM: Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006, 24 (2): 376-385. 10.1634/stemcells.2005-0234.View ArticlePubMed
- Zaragosi LE, Ailhaud G, Dani C: Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells. Stem Cells. 2006, 24 (11): 2412-2419. 10.1634/stemcells.2006-0006.View ArticlePubMed
- Rodriguez AM, Pisani D, Dechesne CA, Turc-Carel C, Kurzenne JY, Wdziekonski B, Villageois A, Bagnis C, Breittmayer JP, Groux H, Ailhaud G, Dani C: Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med. 2005, 201 (9): 1397-1405. 10.1084/jem.20042224.PubMed CentralView ArticlePubMed
- Rodriguez AM, Elabd C, Delteil F, Astier J, Vernochet C, Saint-Marc P, Guesnet J, Guezennec A, Amri EZ, Dani C, Ailhaud G: Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochemical and Biophysical Research Communications. 2004, 315 (2): 255-263. 10.1016/j.bbrc.2004.01.053.View ArticlePubMed
- Elabd C, Chiellini C, Massoudi A, Cochet O, Zaragosi LE, Trojani C, Michiels JF, Weiss P, Carle G, Rochet N, Dechesne CA, Ailhaud G, Dani C, Amri EZ: Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells. Biochem Biophys Res Commun. 2007, 361 (2): 342-348. 10.1016/j.bbrc.2007.06.180.View ArticlePubMed
- Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH: Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004, 10 (1): 55-63. 10.1038/nm979.View ArticlePubMed
- Cho HH, Kim YJ, Kim SJ, Kim JH, Bae YC, Ba B, Jung JS: Endogenous Wnt signaling promotes proliferation and suppresses osteogenic differentiation in human adipose derived stromal cells. Tissue Eng. 2006, 12 (1): 111-121. 10.1089/ten.2006.12.111.View ArticlePubMed
- Nuttall ME, Gimble JM: Controlling the balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications. Curr Opin Pharmacol. 2004, 4 (3): 290-294. 10.1016/j.coph.2004.03.002.View ArticlePubMed
- Kennell JA, MacDougald OA: Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms. J Biol Chem. 2005, 280 (25): 24004-24010. 10.1074/jbc.M501080200.View ArticlePubMed
- Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG, Haydon RC, He TC: Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem. 2004, 279 (53): 55958-55968. 10.1074/jbc.M407810200.View ArticlePubMed
- Chengappa KN, Chalasani L, Brar JS, Parepally H, Houck P, Levine J: Changes in body weight and body mass index among psychiatric patients receiving lithium, valproate, or topiramate: an open-label, nonrandomized chart review. Clin Ther. 2002, 24 (10): 1576-1584. 10.1016/S0149-2918(02)80061-3.View ArticlePubMed
- Wilting I, de Vries F, Thio BM, Cooper C, Heerdink ER, Leufkens HG, Nolen WA, Egberts AC, van Staa TP: Lithium use and the risk of fractures. Bone. 2007, 40 (5): 1252-1258. 10.1016/j.bone.2006.12.055.View ArticlePubMed
- Wdziekonski B, Villageois P, Dani C: Development of adipocytes from differentiated ES cells. Methods Enzymol. 2003, 365: 268-277.View ArticlePubMed
- Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R, Pfeiffer EF: Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium. J Clin Invest. 1989, 84 (5): 1663-1670.PubMed CentralView ArticlePubMed
- Sturn A, Mlecnik B, Pieler R, Rainer J, Truskaller T, Trajanoski Z: Client-server environment for high-performance gene expression data analysis. Bioinformatics. 2003, 19 (6): 772-773. 10.1093/bioinformatics/btg074.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.