Adipogenic differentiation of culture-expanded bone marrow derived porcine mesenchymal stem cells

SUJOY K DHARA; LAKSHMAN SANTRA; SAURABH GUPTA

doi:10.56093/ijans.v87i3.68854

Authors

SUJOY K DHARA ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243 122 India
LAKSHMAN SANTRA ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243 122 India
SAURABH GUPTA ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243 122 India

https://doi.org/10.56093/ijans.v87i3.68854

Keywords:

Adipogenic differentiation, Bone marrow, Porcine MSCs, Obesity model

Abstract

In order to use Mesenchymal stem cell populations for obesity and related metabolic syndrome studies in cell culture system, as a prerequisite, we evaluated the potency of these stem cells to undergo adipogenic differentiation. Porcine stem cells were chosen to study adipogenesis in due to the fact that pig has a natural tendency to get obese and the species is considered to be the most desired biomedical model for human applications. Porcine MSCs have been exposed to adipogenic induction media following a 21day protocol and observed under microscope for detecting stages of differentiation. At the terminal differentiation stage; morphologically, the cells appeared rounded with numerous large cytosolic lipid spheres. Upon staining with Oil Red O, the lipid spheres stained bright red. Based on this, proprietary medium was found to differentiate MSCs more efficiently than medium formulated on previous reports. Both, the differential morphologic feature corresponding to the adipocyte and positive Oil Red O staining confirmed about successful adipogenic differentiation. We envision that stem cell based culture system from porcine species would aid for studying molecular adipogenesis and subsequent identification of therapeutic targets for obesity and other metabolic diseases.

Downloads

Download data is not yet available.

References

Agarwal M. 2013. Expression of transcription factor CRISPLD2 during osteogenic differentiation of pig mesenchymal stem cells. Indian Veterinary Research Institute, Izatnagar, Bareilly. Andres-Manzano M J, Andres V and Dorado B. 2015. Oil Red O and Hematoxylin and Eosin staining for quantification of atherosclerosis burden in mouse aorta and aortic root. Methods in Molecular Biology 1339: 85–99. DOI: https://doi.org/10.1007/978-1-4939-2929-0_5

Boyd N L, Robbins K R, Dhara S K, West F D and Stice S L. 2009. Human embryonic stem cell–derived mesoderm-like epithelium transitions to mesenchymal progenitor cells. Tissue Engineering 15: 1897–1907. DOI: https://doi.org/10.1089/ten.tea.2008.0351

Chakravarthy M V, Pan Z, Zhu Y, Tordjman K, Schneider J G, Coleman T, Turk J and Semenkovich C F. 2005. “New” hepatic fat activates PPAR alpha to maintain glucose, lipid, and cholesterol homeostasis. Cell Metabolism 1: 309–22. DOI: https://doi.org/10.1016/j.cmet.2005.04.002

Crick S J, Sheppard M N, Ho S Y, Gebstein L and Anderson R H. 1998. Anatomy of the pig heart: comparisons with normal human cardiac structure. Journal of Anatomy 193 (Pt 1): 105– 19. DOI: https://doi.org/10.1046/j.1469-7580.1998.19310105.x

Deutsch M J, Schriever S C, Roscher A A and Ensenauer R. 2014. Digital image analysis approach for lipid droplet size quantitation of Oil Red O stained cultured cells. Analytical Biochemistry 445: 87–89. DOI: https://doi.org/10.1016/j.ab.2013.10.001

Esteban M A, Peng M, Deli Z, Cai J, Yang J, Xu J, Lai L and Pei D. 2010. Porcine induced pluripotent stem cells may bridge the gap between mouse and human iPS. IUBMB Life 62: 277– 82. DOI: https://doi.org/10.1002/iub.307

Green C D, Ozguden-Akkoc C G, Wang Y, Jump D B and Olson L K. 2010. Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species. Journal of Lipid Research 51: 1871–77. DOI: https://doi.org/10.1194/jlr.M004747

Green H and Kehinde O. 1975. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5: 19–27. DOI: https://doi.org/10.1016/0092-8674(75)90087-2

Harms M and Seale P. 2013. Brown and beige fat: development, function and therapeutic potential. Nature Medicine 19: 1252– 63. DOI: https://doi.org/10.1038/nm.3361

Jeon T I and Osborne T F. 2012. SREBPs: metabolic integrators in physiology and metabolism. Trends in Endocrinology and Metabolism 23: 65–72. DOI: https://doi.org/10.1016/j.tem.2011.10.004

Jiao F, Wang J, Dong Z L, Wu M J, Zhao T B, Li D D and Wang X. 2012. Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages. Cell Reprogram 14: 324–33. DOI: https://doi.org/10.1089/cell.2012.0004

Joost H G and Schurmann A. 2001. Subcellular fractionation of adipocytes and 3T3-L1 cells. Methods in Molecular Biology 155: 77–82.

Knebel B, Haas J, Hartwig S, Jacob S, Kollmer C, Nitzgen U, Muller-Wieland D and Kotzka J. 2012. Liver-specific expression of transcriptionally active SREBP-1c is associated with fatty liver and increased visceral fat mass. PLoS One 7: e31812. DOI: https://doi.org/10.1371/journal.pone.0031812

Krampera M, Franchini M, Pizzolo G and Aprili G. 2007. Mesenchymal stem cells: from biology to clinical use. Blood Transfusion 5: 120–29.

Kumadaki S, Matsuzaka T, Kato T, Yahagi N, Yamamoto T, Okada S, Kobayashi K, Takahashi A, Yatoh S, Suzuki H, Yamada N and Shimano H. 2008. Mouse Elovl-6 promoter is an SREBP target. Biochemical and Biophysical Research Communications 368: 261–66. DOI: https://doi.org/10.1016/j.bbrc.2008.01.075

Mao J, DeMayo F J, Li H, Abu-Elheiga L, Gu Z, Shaikenov T E, Kordari P, Chirala S S, Heird W C and Wakil S J. 2006. Liver- specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis. Proceedings of the National Academy of Sciences USA 103: 8552–57. DOI: https://doi.org/10.1073/pnas.0603115103

Matsuzaka T and Shimano H. 2009. Elovl6: a new player in fatty acid metabolism and insulin sensitivity. Journal of Molecular Medicine (Berl) 87: 379–84. DOI: https://doi.org/10.1007/s00109-009-0449-0

Matsuzaka T, Shimano H, Yahagi N, Kato T, Atsumi A, Yamamoto T, Inoue N, Ishikawa M, Okada S, Ishigaki N, Iwasaki H, Iwasaki Y, Karasawa T, Kumadaki S, Matsui T, Sekiya M, Ohashi K, Hasty A H, Nakagawa Y, Takahashi A, Suzuki H, Yatoh S, Sone H, Toyoshima H, Osuga J and Yamada N. 2007. Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance. Nature Medicine 13: 1193– 1202. DOI: https://doi.org/10.1038/nm1662

Matsuzaka T, Shimano H, Yahagi N, Yoshikawa T, Amemiya- Kudo M, Hasty A H, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga J, Takahashi A, Yato S, Sone H, Ishibashi S and Yamada N. 2002. Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs. Journal of Lipid Research 43: 911–20. DOI: https://doi.org/10.1016/S0022-2275(20)30465-X

Mehlem A, Hagberg C E, Muhl L, Eriksson U and Falkevall A. 2013. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nature Protocols 8: 1149–54. DOI: https://doi.org/10.1038/nprot.2013.055

Mestas J and Hughes C C. 2004. Of mice and not men: differences between mouse and human immunology. Journal of Immunology 172: 2731–38. DOI: https://doi.org/10.4049/jimmunol.172.5.2731

Miyazaki M, Flowers M T, Sampath H, Chu K, Otzelberger C, Liu X and Ntambi J M. 2007. Hepatic stearoyl-CoA desaturase-1 deficiency protects mice from carbohydrate- induced adiposity and hepatic steatosis. Cell Metabolism 6: 484–96. DOI: https://doi.org/10.1016/j.cmet.2007.10.014

Ntambi J M, Miyazaki M, Stoehr J P, Lan H, Kendziorski C M, Yandell B S, Song Y, Cohen P, Friedman J M and Attie A D. 2002. Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proceedings of the National Academy of Sciences USA 99: 11482–486. DOI: https://doi.org/10.1073/pnas.132384699

Porras A and Santos E.1996. The insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells: dissociation between Raf-1 kinase and the MAPK/RSK cascade. International Journal of Obesity and Related Metabolic Disorders 20 Suppl 3: S43–51.

Samstein B and Platt J L. 2001. Physiologic and immunologic hurdles to xenotransplantation. Journal of American Society of Nephrology 12: 182–93. DOI: https://doi.org/10.1681/ASN.V121182

Santra L. 2016. Epigenetic regulation of CRISPLD2 gene during osteogenic differentiation of porcine bone marrow derived mesenchymal stem cells. Indian Veterinary Research Institute, Izatnagar, Bareilly.

Santra L, Gupta S, Kannan S, Singh A K, Ravi Kumar G, Naskar S, Ghosh J and Dhara S K. 2017. Long bones, a slaughterhouse by-product, may serve as an excellent source for mesenchymal stem cells. Indian Journal of Animal Sciences 87(1): 53–58. DOI: https://doi.org/10.56093/ijans.v87i1.66860

Santra L, Gupta S, Singh A K, Sahu A R, Gandham R K, Naskar S, Maity S K, Ghosh J and Dhara S K. 2015. A comparative analysis of invasive and non-invasive method of bone marrow stromal cell isolation. Asian Journal of Animal and Veterinary Advances 10: 549–55. DOI: https://doi.org/10.3923/ajava.2015.549.555

Shimano H. 2012. Novel qualitative aspects of tissue fatty acids related to metabolic regulation: lessons from Elovl6 knockout. Progress in Lipid Research 51: 267–71. DOI: https://doi.org/10.1016/j.plipres.2011.12.004

Swindle M M, Makin A, Herron A J, Clubb F J Jr. and Frazier K S. 2012. Swine as models in biomedical research and toxicology testing. Veterinary Pathology 49: 344–56. DOI: https://doi.org/10.1177/0300985811402846

Ye L, Chang Y H, Xiong Q, Zhang P, Zhang L, Somasundaram P, Lepley M, Swingen C, Su L, Wendel J S, Guo J, Jang A, Rosenbush D, Greder L, Dutton J R, Zhang J, Kamp T J, Kaufman D S, Ge Y and Zhang J. 2014. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell 15: 750–61. DOI: https://doi.org/10.1016/j.stem.2014.11.009