Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids

C-L Wu, B O Diekman, D Jain, F Guilak, C-L Wu, B O Diekman, D Jain, F Guilak

Abstract

Introduction: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues.

Materials and methods: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro.

Results: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells.

Conclusions: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Conflict of interest statement

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors have nothing to disclose

Figures

Figure 1
Figure 1
Representative stem cell sorting result of (A–C) bone marrow and (D–F) inguinal fat from lean mice. In the bone marrow of lean mice, approximately 1% of stromal cells were Ter119/CD45 double-negative (green dots). Only 5% of these cells were Sca-1/PDGFRα double-positive (red dots) and the cells that were Sca-1+ PDGFRα+ Ter119− CD45− were identified as MSCs. In inguinal fat of lean mice, around 24% of adipose-tissue stromal cells were CD31/Ter119/CD45 triple-negative (green dots). Among this population, 63% were Sca-1/CD34 double-positive (red dots) and sqASCs were designated as the cells that were Sca-1+ CD34+ CD31− Ter119− CD45−. Obese mice showed a trend toward increased in vivo frequency of (B) MSCs (n = 5 isolations) and (E) sqASCs (n = 3 isolations). n ≥ 4 mice per isolation. In the stem cell population, obesity significantly increased (C) the percentage of Sca-1+ PDGFRα+ cells (red bar) among Ter119− CD45− cell population (green bar) in the bone marrow (# p < 0.05 vs. corresponding lean cell population). (F) Obese mice showed a trend toward increased CD45− CD31− Ter119− cells (green bar) in the inguinal fat.
Figure 2
Figure 2
Morphology of (A, C, E) lean and (B, D, F) obese stem cells at passage 3. The cumulative fold increase during expansion under hypoxic conditions through passage 5 of (G) MSCs (H) sqASCs and (I) IFP cells harvested from lean and obese mice. Obese sqASCs and IFP cells showed a trend toward increased proliferation, while obese MSCs had a trend toward decreased cell growth. Results averaged from 3 independent isolations with mean ± SEM displayed (n ≥ 3 mice per isolation). Scale bar is 100 μm.
Figure 3
Figure 3
Adipogenesis and osteogenesis of stem cells harvested from lean and obese mice. Lipid droplets accumulation in (A, G, M) lean and (B, H, N) obese stem cells after 14 days culture in adipogenic medium. Cells were stained with 0.5% Oil Red O. Stain was then released and normalized to DNA content to quantify adipogenic potential of (E) MSCs, (K) sqASCs and (Q) IFP cells. For osteogenesis, calcium mineral deposits stained with 2% Alizarin Red S in (C, I, O) lean and (D, J, P) obese stem cells after 21 days culture in osteogenic medium. Stain was then extracted and normalized to DNA content to determine osteogenic capacity of (F) MSCs, (L) sqASCs and (R) IFP cells. Results from≥ 5 samples per group of the cells pooled from two independent isolations (n = 6 mice per isolation) with mean ± SEM displayed. # p < 0.05 vs. corresponding lean cell type by t-test. Scale bar is 100 μm for adipo- and 5 mm for osteogenesis, respectively.
Figure 4
Figure 4
Sulfated GAGs and proteoglycans of chondrogenic pellets from (A, F, K) lean stem cells and (B, G, L) obese stem cells after 28 days pellet culture in chondrogenic medium was detected by 1% Alcian Blue staining (pH = 1). Collagen II immunohistochemical staining was also performed for the pellets from (C, H, M) lean stem cells and (D, I, N) obese stem cells. Quantification of GAG content was performed by DMB assay and the value was then further normalized to DNA to determine chondrogenic potential of (E) MSCs, (J) sqASCs and (O) IFP cells. Obese MSCs exhibited a trend toward decreased chondrogenesis (p = 0.07 vs. lean MSCs), while obese sqASCs and IFP cells showed significantly decreased chondrogenic capacity. Results from ≥ 4 pellets per group of the cells pooled from two independent isolations (n = 6 mice per isolation) with mean ± SEM displayed. # p < 0.05 vs. corresponding lean cell type by t-test. Scale bar is 500 μm.
Figure 5
Figure 5
Multi-lineage differentiation of lean stem cells with supplement of SA/MUFA. (A, D, G) all the stem cells treated with SA/MUFA demonstrated increased adipogenesis. SA/MUFA also significantly enhanced (B) osteogenesis of MSCs but did not significantly affect (E, H) osteogenic potential of sqASCs and IFP cells. However, MSCs did not alter (C) chondrogenic potential in response to SA/MUFA but the treatment of SA/MUFA significantly decreased (F, I) chondrogenic capacity of sqASCs and IFP cells. Results from 5 samples (for adipogenesis and osteogenesis) or ≥ 4 pellets (for chondrogenesis) per group of the cells pooled from two independent isolations (n = 6 mice per isolation) with mean ± SEM displayed. # p < 0.05 vs. vehicle control by t-test.

References

    1. Greenberg AS, Coleman RA, Kraemer FB, McManaman JL, Obin MS, Puri V, et al. The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest. 2011;121(6):2102–2110.
    1. Griffin TM, Fermor B, Huebner JL, Kraus VB, Rodriguiz RM, Wetsel WC, et al. Diet-induced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice. Arthritis Res Ther. 2010;12 (4):R130.
    1. Kuo JH, Wong MS, Perez RV, Li CS, Lin TC, Troppmann C. Renal Transplant Wound Complications in the Modern Era of Obesity. J Surg Res. 2011;173(2):216–223.
    1. Bredella MA, Torriani M, Ghomi RH, Thomas BJ, Brick DJ, Gerweck AV, et al. Vertebral bone marrow fat is positively associated with visceral fat and inversely associated with IGF-1 in obese women. Obesity (Silver Spring) 2011;19(1):49–53.
    1. Bechmann LP, Hannivoort RA, Gerken G, Hotamisligil GS, Trauner M, Canbay A. The interaction of hepatic lipid and glucose metabolism in liver diseases. J Hepatol. 2012;56(4):952–964.
    1. Thanassoulis G, Massaro JM, Hoffmann U, Mahabadi AA, Vasan RS, O’Donnell CJ, et al. Prevalence, distribution, and risk factor correlates of high pericardial and intrathoracic fat depots in the Framingham heart study. Circ Cardiovasc Imaging. 2010;3(5):559–566.
    1. McGuire TR, Brusnahan SK, Bilek LD, Jackson JD, Kessinger MA, Berger AM, et al. Inflammation associated with obesity: relationship with blood and bone marrow endothelial cells. Obesity (Silver Spring) 2011;19(11):2130–2136.
    1. Wang C, Seifert RA, Bowen-Pope DF, Kregel KC, Dunnwald M, Schatteman GC. Diabetes and aging alter bone marrow contributions to tissue maintenance. Int J Physiol Pathophysiol Pharmacol. 2009;2(1):20–28.
    1. Lumeng CN, DelProposto JB, Westcott DJ, Saltiel AR. Phenotypic Switching of Adipose Tissue Macrophages With Obesity Is Generated by Spatiotemporal Differences in Macrophage Subtypes. Diabetes. 2008;57(12):3239–3246.
    1. Nguyen MTA, Favelyukis S, Nguyen AK, Reichart D, Scott PA, Jenn A, et al. A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via toll-like receptors 2 and 4 and JNK-dependent pathways. Journal of Biological Chemistry. 2007;282(48):35279–35292.
    1. Hirabara SM, Curi R, Maechler P. Saturated fatty acid-induced insulin resistance is associated with mitochondrial dysfunction in skeletal muscle cells. J Cell Physiol. 2010;222(1):187–194.
    1. Kruger MC, Coetzee M, Haag M, Weiler H. Long-chain polyunsaturated fatty acids: selected mechanisms of action on bone. Prog Lipid Res. 2010;49(4):438–449.
    1. Lippiello L, Walsh T, Fienhold M. The association of lipid abnormalities with tissue pathology in human osteoarthritic articular cartilage. Metabolism. 1991;40 (6):571–576.
    1. Noth U, Osyczka AM, Tuli R, Hickok NJ, Danielson KG, Tuan RS. Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res. 2002;20(5):1060–1069.
    1. Rosen CJ, Bouxsein ML. Mechanisms of disease: is osteoporosis the obesity of bone? Nat Clin Pract Rheum. 2006;2(1):35–43.
    1. Fujisaka S, Usui I, Bukhari A, Ikutani M, Oya T, Kanatani Y, et al. Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice. Diabetes. 2009;58(11):2574–82.
    1. Wickham MQ, Erickson GR, Gimble JM, Vail TP, Guilak F. Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin Orthop Relat Res. 2003;412:196–212.
    1. Roldan M, Macias-Gonzalez M, Garcia R, Tinahones FJ, Martin M. Obesity short-circuits stemness gene network in human adipose multipotent stem cells. FASEB J. 2011;25(12):4111–4126.
    1. Lv S, Wu L, Cheng P, Yu J, Zhang AS, Zha JM, et al. Correlation of obesity and osteoporosis: Effect of free fatty acids on bone marrow-derived mesenchymal stem cell differentiation. Exp Ther Med. 2010;1(4):603–610.
    1. Tallman DL, Taylor CG. Effects of dietary fat and zinc on adiposity, serum leptin and adipose fatty acid composition in C57BL/6J mice. J Nutr Biochem. 2003;14(1):17–23.
    1. Kleemann R, van Erk M, Verschuren L, van den Hoek AM, Koek M, Wielinga PY, et al. Time-Resolved and Tissue-Specific Systems Analysis of the Pathogenesis of Insulin Resistance. PLoS ONE. 2010;5(1):e8817.
    1. Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc. 2010;5(7):1294–1311.
    1. Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med. 2009;206(11):2483–2496.
    1. Diekman BO, Wu CL, Louer CR, Furman BD, Huebner JL, Kraus VB, et al. Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents post-traumatic arthritis. Cell Transplant. (in press)
    1. Rodeheffer MS, Birsoy K, Friedman JM. Identification of White Adipocyte Progenitor Cells In Vivo. Cell. 2008;135(2):240–249.
    1. Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004;329(1):77–84.
    1. Liang SX, Khachigian LM, Ahmadi Z, Yang M, Liu S, Chong BH. In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side-population cells) J Thromb Haemost. 2011;9(8):1628–1637.
    1. Maumus M, Peyrafitte JA, D’Angelo R, Fournier-Wirth C, Bouloumie A, Casteilla L, et al. Native human adipose stromal cells: localization, morphology and phenotype. Int J Obes (Lond) 2011;35(9):1141–1153.
    1. Maumus M, Sengenes C, Decaunes P, Zakaroff-Girard A, Bourlier V, Lafontan M, et al. Evidence of in Situ Proliferation of Adult Adipose Tissue-Derived Progenitor Cells: Influence of Fat Mass Microenvironment and Growth. J Clin Endocr Metab. 2008;93(10):4098–4106.
    1. Joe AWB, Yi L, Even Y, Vogl AW, Rossi FMV. Depot-Specific Differences in Adipogenic Progenitor Abundance and Proliferative Response to High-Fat Diet. Stem Cells. 2009;27(10):2563–2570.
    1. Fernandez M, Acuna MJ, Reyes M, Olivares D, Hirsch S, Bunout D, et al. Proliferation and differentiation of human adipocyte precursor cells: differences between the preperitoneal and subcutaneous compartments. J Cel Biochem. 2010;111(3):659–664.
    1. Baptista LS, Silva KR, Pedrosa CSG, Claudio-da-Silva C, Carneiro JRI, Aniceto M, et al. Adipose Tissue of Control and Ex-Obese Patients Exhibit Differences in Blood Vessel Content and Resident Mesenchymal Stem Cell Population. Obes Surg. 2009;19(9):1304–1312.
    1. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008;3(3):301–313.
    1. Caplan AI. Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng. 2005;11(7–8):1198–1211.
    1. Cao JJ. Effects of obesity on bone metabolism. J Orthop Surg Res. 2011;6:30.
    1. Chen JR, Lazarenko OP, Wu X, Tong Y, Blackburn ML, Shankar K, et al. Obesity reduces bone density associated with activation of PPARgamma and suppression of Wnt/beta-catenin in rapidly growing male rats. PLoS ONE. 2010;5(10):e13704.
    1. Majka SM, Barak Y, Klemm DJ. Concise review: adipocyte origins: weighing the possibilities. Stem Cells. 2011;29(7):1034–1040.
    1. Ibrahim MM. Subcutaneous and visceral adipose tissue: structural and functional differences. Obes Rev. 2010;11(1):11–18.
    1. Otabe S, Yuan X, Fukutani T, Wada N, Hashinaga T, Nakayama H, et al. Overexpression of human adiponectin in transgenic mice results in suppression of fat accumulation and prevention of premature death by high-calorie diet. Am J Physiol Endocrinol Metab. 2007;293(1):E210–218.
    1. Ling L, Nurcombe V, Cool SM. Wnt signaling controls the fate of mesenchymal stem cells. Gene. 2009;433(1–2):1–7.
    1. Luzi E, Marini F, Sala SC, Tognarini I, Galli G, Brandi ML. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res. 2008;23 (2):287–295.
    1. Li Z, Hassan MQ, Volinia S, van Wijnen AJ, Stein JL, Croce CM, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A. 2008;105(37):13906–13911.
    1. Lutzner J, Kasten P, Gunther KP, Kirschner S. Surgical options for patients with osteoarthritis of the knee. Nat Rev Rheumatol. 2009;5(6):309–316.
    1. Guilak F, Estes BT, Diekman BO, Moutos FT, Gimble JM. 2010 Nicolas Andry Award: Multipotent adult stem cells from adipose tissue for musculoskeletal tissue engineering. Clin Orthop Relat Res. 2010;468(9):2530–2540.
    1. Hwa Cho H, Bae YC, Jung JS. Role of toll-like receptors on human adipose-derived stromal cells. Stem Cells. 2006;24(12):2744–2752.
    1. Dallas NA, Samuel S, Xia L, Fan F, Gray MJ, Lim SJ, et al. Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy. Clin Cancer Res. 2008;14(7):1931–1937.
    1. Quintana L, zur Nieden NI, Semino CE. Morphogenetic and regulatory mechanisms during developmental chondrogenesis: new paradigms for cartilage tissue engineering. Tissue Eng Part B Rev. 2009;15(1):29–41.
    1. Hellingman CA, Davidson EN, Koevoet W, Vitters EL, van den Berg WB, van Osch GJ, et al. Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification. Tissue Eng Part A. 2011;17(7–8):1157–1167.
    1. Ataliotis P. Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro. Mech Develop. 2000;94(1–2):13–24.
    1. Koay EJ, Athanasiou KA. Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality. Osteoarthr Cartilage. 2008;16(12):1450–1456.
    1. Deshimaru R, Ishitani K, Makita K, Horiguchi F, Nozawa S. Analysis of fatty acid composition in human bone marrow aspirates. Keio J Med. 2005;54(3):150–155.
    1. Yu S, Cho HH, Joo HJ, Bae YC, Jung JS. Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells. Mol Cell Biochem. 2008;317(1–2):143–150.
    1. Pevsner-Fischer M, Morad V, Cohen-Sfady M, Rousso-Noori L, Zanin-Zhorov A, Cohen S, et al. Toll-like receptors and their ligands control mesenchymal stem cell functions. Blood. 2007;109(4):1422–1432.
    1. Mo IF, Yip KH, Chan WK, Law HK, Lau YL, Chan GC. Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors. BMC Cell Biol. 2008;9:52.
    1. German JB, Dillard CJ. Saturated fats: what dietary intake? Am J Clin Nutr. 2004;80(3):550–559.
    1. Manickam E, Sinclair AJ, Cameron-Smith D. Suppressive actions of eicosapentaenoic acid on lipid droplet formation in 3T3-L1 adipocytes. Lipids Health Dis. 2010;9:57.
    1. Scheller EL, Song J, Dishowitz MI, Soki FN, Hankenson KD, Krebsbach PH. Leptin functions peripherally to regulate differentiation of mesenchymal progenitor cells. Stem Cells. 2010;28(6):1071–1080.
    1. Zhao L, Huang J, Zhang H, Wang Y, Matesic LE, Takahata M, et al. Tumor necrosis factor inhibits mesenchymal stem cell differentiation into osteoblasts via the ubiquitin E3 ligase Wwp1. Stem Cells. 2011;29(10):1601–1610.
    1. Shin JH, Shin DW, Noh M. Interleukin-17A inhibits adipocyte differentiation in human mesenchymal stem cells and regulates pro-inflammatory responses in adipocytes. Biochem Pharmacol. 2009;77(12):1835–1844.
    1. Noth U, Rackwitz L, Steinert AF, Tuan RS. Cell delivery therapeutics for musculoskeletal regeneration. Adv Drug Deliv Rev. 2010;62(7–8):765–783.

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