Abstract
Fetal bovine serum (FBS), which is widely used in cell culture media, has the potential to cause medical and ethical problems. Here, an experimental study using milk or whey proteins containing essential nutrients and growth factors is presented to limit the use of FBS in cell culture media produced for cell and tissue regeneration. Study groups were formed by culturing human placenta mesenchymal stem cells, known to have high proliferation and differentiation capacity, with milk or whey solution at increasing concentrations, alone or in combination with FBS. Osteogenic and adipogenic differentiation capacities of proliferating cells were observed in FBS, milk or whey groups. Milk, whey or FBS groups obtained in P3 and after differentiation were separately analyzed for protein mRNA expression by reverse transcriptase-polymerase chain reaction (RT-qPCR). Fibroblast Growth Factor 2 (FGF2), Octamer-binding Transcription Factor 4 (OCT4), Bone Morphogenetic Protein 6 (BMP6), and adipogenic differentiation marker Peroxisome Proliferator-Activated Receptor Gamma (PPARG) were analysed by RT-qPCR. Proliferation was more pronounced in FBS alone and in its combinations with milk-whey compared to the groups in which only milk and whey were used. OCT4 mRNA and FGF2 mRNA expression decreased in differentiated cells. BMP6 mRNA expression increased with osteogenic and adipogenic stimuli. As expected, PPRG expression also increased with adipogenic stimulation. With this experimental study, evidence has been obtained that milk or whey can provide nutritional support to the culture media of repair cells and preserve the functional capacity of the cells, with a slightly more limited capacity than FBS.
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References
Almeida CC, Alvares TS, Costa MP, Conte-Junior CA (2016) Protein and amino acid profiles of different whey protein supplements. J Diet Suppl 13:313–323. https://doi.org/10.3109/19390211.2015.1036187
Beeravolu N, McKee C, Alamri A, Mikhael S, Brown C, Perez-Cruet M, Chaudhry GR (2017) Isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta. J Vis Exp 122:55224. https://doi.org/10.3791/55224
Berebichez-Fridman R, Montero-Olvera PR (2018) Sources and clinical applications of mesenchymal stem cells: state-of-the-art review. Sultan Qaboos Univ Med J 18:e264–e277. https://doi.org/10.18295/squmj.2018.18.03.002
Carreira AC, Zambuzzi WF, Rossi MC, Astorino Filho R, Sogayar MC, Granjeiro JM (2015) Bone morphogenetic proteins: promising molecules for bone healing, bioengineering, and regenerative medicine. Vitam Horm 99:293–322. https://doi.org/10.1016/bs.vh.2015.06.002
Chetta KE, Alcorn JL, Baatz JE, Wagner CL (2021) Cytotoxic lactalbumin-oleic acid complexes in the human milk Diet of Preterm Infants. Nutrients 13:4336. https://doi.org/10.3390/nu13124336
D’Souza K, Mercer A, Mawhinney H, Pulinilkunnil T, Udenigwe CC, Kienesberger PC (2020) Whey peptides stimulate differentiation and lipid metabolism in adipocytes and ameliorate lipotoxicity-induced insulin resistance in muscle cells. Nutrients 12:425. https://doi.org/10.3390/nu12020425
Dev K, Giri SK, Kumar A, Yadav A, Singh B, Gautam SK (2012) Expression of transcriptional factor genes (Oct-4, nanog, and Sox-2) and embryonic stem cell-like characters in placental membrane of Buffalo (Bubalus bubalis). J Membr Biol 245:177–183. https://doi.org/10.1007/s00232-012-9427-5
Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A (2005) Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells 23:1200–1211. https://doi.org/10.1634/stemcells.2004-0303
Electricwala A (1992) Can milk replace serum in mammalian cell culture? Cytotechnology 8:1–4. https://doi.org/10.1007/BF02540023
Gilliland G, Perrin S, Bunn HF (1990) Competitive PCR for quantitation of mRNA. In: Innis MA (ed) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 60–69
Grafe I, Alexander S, Peterson JR, Snider TN, Levi B, Lee B, Mishina Y (2018) TGF-β family signaling in mesenchymal differentiation. Cold Spring Harb Perspect Biol 10:a022202. https://doi.org/10.1101/cshperspect.a022202
Hassiotou F, Beltran A, Chetwynd E, Stuebe AM, Twigger AJ, Metzger P, Trengove N, Lai CT, Filgueira L, Blancafort P, Hartmann PE (2012) Breastmilk is a novel source of stem cells with multilineage differentiation potential. Stem Cells 30:2164–2174. https://doi.org/10.1002/stem.1188
Hassiotou F, Geddes DT, Hartmann PE (2013) Cells in human milk: state of the science. J Hum Lact 29:171–182. https://doi.org/10.1177/0890334413477242
Karlsson H, Erkers T, Nava S, Ruhm S, Westgren M, Ringdén O (2012) Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro. Clin Exp Immunol 167:543–555. https://doi.org/10.1111/j.1365-2249.2011.04540.x
Kmiecik G, Niklińska W, Kuć P, Pancewicz-Wojtkiewicz J, Fil D, Karwowska A, Karczewski J, Mackiewicz Z (2013) Fetal membranes as a source of stem cells. Adv Med Sci 58:185–195. https://doi.org/10.2478/ams-2013-0007
Kozanoglu I, Maytalman E (2012) Isolation and identification of mesenchymal stem cells. In: Turksen K (ed) Adult and embryonic stem cells, 1st edn. Humana Press, New York, pp 25–32
Ma S, Xie N, Li W, Yuan B, Shi Y, Wang Y (2014) Immunobiology of mesenchymal stem cells. Cell Death Differ 21:216–225. https://doi.org/10.1038/cdd.2013.158
Marongiu F, Gramignoli R, Sun Q, Tahan V, Miki T, Dorko K, Ellis E, Strom SC (2010) Isolation of amniotic mesenchymal stem cells. Curr Protoc Stem Cell Biol. https://doi.org/10.1002/9780470151808.sc01e05s12
Maytalman E, Alizadeh Yegani A, Kozanoglu I, Aksu F (2022) Adrenergic receptor behaviors of mesenchymal stem cells obtained from different tissue sources and the effect of the receptor blockade on differentiation. J Recept Signal Transduct Res 42:349–360. https://doi.org/10.1080/10799893.2021.1957931
Muruganandan S, Roman AA, Sinal CJ (2009) Adipocyte differentiation of bone marrow-derived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 66:236–253. https://doi.org/10.1007/s00018-008-8429-z
Patki S, Kadam S, Chandra V, Bhonde R (2010) Human breast milk is a rich source of multipotent mesenchymal stem cells. Hum Cell 23:35–40. https://doi.org/10.1111/j.1749-0774.2010.00083.x
Präbst K, Engelhardt H, Ringgeler S, Hübner H (2017) Basic colorimetric proliferation assays: MTT, WST, and resazurin. Methods Mol Biol 1601:1–17. https://doi.org/10.1007/978-1-4939-6960-9_1
Redondo LM, García V, Peral B, Verrier A, Becerra J, Sánchez A, García-Sancho J (2018) Repair of maxillary cystic bone defects with mesenchymal stem cells seeded on a cross-linked serum scaffold. J Craniomaxillofac Surg 46:222–229. https://doi.org/10.1016/j.jcms.2017.11.004
Rushkevich YN, Kosmacheva SM, Zabrodets GV, Ignatenko SI, Goncharova NV, Severin IN, Likhachev SA, Potapnev MP (2015) The use of autologous mesenchymal stem cells for cell therapy of patients with amyotrophic lateral sclerosis in Belarus. Bull Exp Biol Med 159:576–581. https://doi.org/10.1007/s10517-015-3017-3
Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A, Wengler GS, Parolini O (2007) Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 1:296–305. https://doi.org/10.1002/term.40
Squillaro T, Peluso G, Galderisi U (2016) Clinical trials with mesenchymal stem cells: an update. Cell Transpl 25:829–848. https://doi.org/10.3727/096368915X689622
Tekkatte C, Gunasingh GP, Cherian KM, Sankaranarayanan K (2011) Humanized” stem cell culture techniques: the animal serum controversy. Stem Cells Int 2011:504723. https://doi.org/10.4061/2011/504723
Tonarova P, Lochovska K, Pytlik R, Hubalek Kalbacova M (2021) The impact of various culture conditions on human mesenchymal stromal cells metabolism. Stem Cells Int 2021:6659244. https://doi.org/10.1155/2021/6659244
Xu R (2009) Effect of whey protein on the proliferation and differentiation of osteoblasts. J Dairy Sci 92:3014–3018. https://doi.org/10.3168/jds.2008-1702
Yang N, Qian S, Jiang Z, Hou J (2021) Cysteine inducing formation and reshuffling of disulfide bonds in cold-extruded whey protein molecules: from structural and functional characteristics to cytotoxicity. Food Chem 360:130121. https://doi.org/10.1016/j.foodchem.2021.130121
Yuan Z, Li Q, Luo S, Liu Z, Luo D, Zhang B, Zhang D, Rao P, Xiao J (2016) PPARγ and wnt signaling in adipogenic and osteogenic differentiation of mesenchymal stem cells. Curr Stem Cell Res Ther 11:216–225. https://doi.org/10.2174/1574888x10666150519093429
Yuan E, Zhou M, Nie S, Ren J (2022) Interaction mechanism between ZnO nanoparticles-whey protein and its effect on toxicity in GES-1 cells. J Food Sci 87:2417–2426. https://doi.org/10.1111/1750-3841.16193
Acknowledgements
We would like to thank the managers of Başkent University Adana Teaching and Medical Research Center & Hematology Research Laboratory, who supported our study. We also thank the Turkish Scientific and Technological Research Council (TÜBİTAK) for encouraging us to carry out our study. In addition, we also thank Dilara Nemutlu Samur for her support for spell checks.
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The project idea was put forward by BB and MA. BB, MA, and EM jointly designed and conducted the laboratory phases of the study. IK provided laboratory support. The writing phase of the study was done by all the authors.
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Boga, B., Akbulut, M., Maytalman, E. et al. Effect of milk and whey on proliferation and differentiation of placental stromal cells. Cytotechnology 75, 391–401 (2023). https://doi.org/10.1007/s10616-023-00585-z
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DOI: https://doi.org/10.1007/s10616-023-00585-z