In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.
Abstract: The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O, 5% O, or 20% to 5% O) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes , , and ; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O and 20% to 5% O), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O presented a more compact morphology with a well-defined border than cells cultured in 5% O and 20% to 5% O. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (, , , , and ), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.
Copyright © 2020 Raquel V. G. de Castro et al.
Publication Date: 2020-11-26 PubMed ID: 33456472PubMed Central: PMC7785345DOI: 10.1155/2020/8814989Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article examines the effect of different oxygen levels on the successful transformation of horse fibroblasts into pluripotent stem cells. It finds that lowered oxygen tension does not benefit the stem cell’s pluripotency and that culture conditions, including oxygen atmosphere, should be considered to maintain pluripotency.
Objective and Approach
- The research aimed to derive and maintain pluripotent stem cells (abbreviated as eiPSCs) from an old horse fibroblasts (the horse was over 20 years) and assess how varying degrees of oxygen tension (atmospheric 20% O, 5% O, or a variable 20% to 5% O) affect the cells.
- Fibroblasts were artificially manipulated (reprogrammed) into becoming eiPSCs.
Observations
- The resulting eiPSCs tested positive for alkaline phosphatase, indicating cell pluripotency, along with other pluripotency-related genes.
- Immunofluorescence-positive staining was detected for OCT4 and NANOG for all groups; SOX2 for 5% O and 20% to 5% O groups; and TRA-1-60, TRA-1-81, and SSEA-1 only in the 20% O group.
- The eiPSCs could form embryoid bodies and differentiate across mesoderm, endoderm, and ectoderm embryonic germ layers.
Findings and Conclusion
- eiPSC colonies grown in 20% O appeared more compact with well-defined borders compared to those in 5% O and fluctuating 20% to 5% O.
- Differences were found in the expression of genes connected to glucose metabolism, mitochondrial division, and hypoxia in response to reprogramming.
- The research found no hindrance to eiPSCs derivation due to aging, suggesting that even mature cells can be reprogrammed into pluripotent stem cells.
- Lower oxygen tension did not favor the pluripotency of eiPSCs. In essence, the atmospheric oxygen level under which eiPSCs are cultured plays a pivotal role in their pluripotency capabilities.
Cite This Article
APA
de Castro RVG, Pieri NCG, Fantinato Neto P, Grizendi BM, Du00f3ria RGS, Meirelles FV, Smith LC, Garcia JM, Bressan FF.
(2020).
In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.
Stem Cells Int, 2020, 8814989.
https://doi.org/10.1155/2020/8814989 Publication
Researcher Affiliations
- Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, Su00e3o Paulo State University, 14884-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Su00e3o Paulo (FMVZ/USP), 13635-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
- Centre de Recherche en Reproduction et Fertilitu00e9, Facultu00e9 de Mu00e9decine Vu00e9tu00e9rinaire, Universitu00e9 de Montru00e9al, QC, Canada J2S 2M2.
- Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, Su00e3o Paulo State University, 14884-900, Brazil.
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Su00e3o Paulo (FZEA/USP), 13635-900, Brazil.
Conflict of Interest Statement
The authors declare that there is no conflict of interest regarding the publication of this paper.
References
This article includes 59 references
- Doss MX, Sachinidis A. Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications.. Cells 2019 Apr 30;8(5).
- Koh S, Piedrahita JA. From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals.. Theriogenology 2014 Jan 1;81(1):103-11.
- Volk SW, Theoret C. Translating stem cell therapies: the role of companion animals in regenerative medicine.. Wound Repair Regen 2013 May-Jun;21(3):382-94.
- Smith RK, Garvican ER, Fortier LA. The current 'state of play' of regenerative medicine in horses: what the horse can tell the human.. Regen Med 2014;9(5):673-85.
- Cong X, Zhang SM, Ellis MW, Luo J. Large Animal Models for the Clinical Application of Human Induced Pluripotent Stem Cells.. Stem Cells Dev 2019 Oct 1;28(19):1288-1298.
- Pessu00f4a LVF, Bressan FF, Freude KK. Induced pluripotent stem cells throughout the animal kingdom: Availability and applications.. World J Stem Cells 2019 Aug 26;11(8):491-505.
- McGowan TW, Pinchbeck G, Phillips CJ, Perkins N, Hodgson DR, McGowan CM. A survey of aged horses in Queensland, Australia. Part 2: Clinical signs and owners' perceptions of health and welfare.. Aust Vet J 2010 Dec;88(12):465-71.
- Sage AM. Cardiac disease in the geriatric horse.. Vet Clin North Am Equine Pract 2002 Dec;18(3):575-89, viii.
- Ireland JL, Clegg PD, McGowan CM, McKane SA, Chandler KJ, Pinchbeck GL. Disease prevalence in geriatric horses in the United Kingdom: veterinary clinical assessment of 200 cases.. Equine Vet J 2012 Jan;44(1):101-6.
- Brosnahan MM, Paradis MR. Demographic and clinical characteristics of geriatric horses: 467 cases (1989-1999).. J Am Vet Med Assoc 2003 Jul 1;223(1):93-8.
- Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence.. Genes Dev 2010 Nov 15;24(22):2463-79.
- Wiley CD, Velarde MC, Lecot P, Liu S, Sarnoski EA, Freund A, Shirakawa K, Lim HW, Davis SS, Ramanathan A, Gerencser AA, Verdin E, Campisi J. Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype.. Cell Metab 2016 Feb 9;23(2):303-14.
- Trokovic R, Weltner J, Noisa P, Raivio T, Otonkoski T. Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells.. Stem Cell Res 2015 Jul;15(1):254-62.
- Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, Hochedlinger K. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.. Nature 2009 Aug 27;460(7259):1145-8.
- Mahmoudi S, Mancini E, Xu L, Moore A, Jahanbani F, Hebestreit K, Srinivasan R, Li X, Devarajan K, Pru00e9lot L, Ang CE, Shibuya Y, Benayoun BA, Chang ALS, Wernig M, Wysocka J, Longaker MT, Snyder MP, Brunet A. Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing.. Nature 2019 Oct;574(7779):553-558.
- Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. Hypoxia enhances the generation of induced pluripotent stem cells.. Cell Stem Cell 2009 Sep 4;5(3):237-41.
- Sperber H, Mathieu J, Wang Y, Ferreccio A, Hesson J, Xu Z, Fischer KA, Devi A, Detraux D, Gu H, Battle SL, Showalter M, Valensisi C, Bielas JH, Ericson NG, Margaretha L, Robitaille AM, Margineantu D, Fiehn O, Hockenbery D, Blau CA, Raftery D, Margolin AA, Hawkins RD, Moon RT, Ware CB, Ruohola-Baker H. The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.. Nat Cell Biol 2015 Dec;17(12):1523-35.
- Mathieu J, Zhang Z, Nelson A, Lamba DA, Reh TA, Ware C, Ruohola-Baker H. Hypoxia induces re-entry of committed cells into pluripotency.. Stem Cells 2013 Sep;31(9):1737-48.
- Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.. Nat Cell Biol 2003 Aug;5(8):741-7.
- Wu J, Ocampo A, Belmonte JCI. Cellular Metabolism and Induced Pluripotency.. Cell 2016 Sep 8;166(6):1371-1385.
- DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation.. Cell Metab 2008 Jan;7(1):11-20.
- Nishimura K, Fukuda A, Hisatake K. Mechanisms of the Metabolic Shift during Somatic Cell Reprogramming.. Int J Mol Sci 2019 May 7;20(9).
- Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation.. Science 2009 May 22;324(5930):1029-33.
- Pessu00f4a LVF, Pires PRL, Del Collado M, Pieri NCG, Recchia K, Souza AF, Perecin F, da Silveira JC, de Andrade AFC, Ambrosio CE, Bressan FF, Meirelles FV. Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues.. Stem Cells Int 2019;2019:1393791.
- Bressan FF, Bassanezze V, de Figueiredo Pessu00f4a LV, Sacramento CB, Malta TM, Kashima S, Fantinato Neto P, Strefezzi RF, Pieri NCG, Krieger JE, Covas DT, Meirelles FV. Generation of induced pluripotent stem cells from large domestic animals.. Stem Cell Res Ther 2020 Jun 25;11(1):247.
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.. Methods 2001 Dec;25(4):402-8.
- Djojosubroto MW, Choi YS, Lee HW, Rudolph KL. Telomeres and telomerase in aging, regeneration and cancer.. Mol Cells 2003 Apr 30;15(2):164-75.
- Allsopp RC, Chang E, Kashefi-Aazam M, Rogaev EI, Piatyszek MA, Shay JW, Harley CB. Telomere shortening is associated with cell division in vitro and in vivo.. Exp Cell Res 1995 Sep;220(1):194-200.
- Marion RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, Serrano M, Blasco MA. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.. Cell Stem Cell 2009 Feb 6;4(2):141-54.
- Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells.. Nat Rev Mol Cell Biol 2007 Sep;8(9):729-40.
- Zhao R, Choi BY, Lee MH, Bode AM, Dong Z. Implications of Genetic and Epigenetic Alterations of CDKN2A (p16(INK4a)) in Cancer.. EBioMedicine 2016 Jun;8:30-39.
- Deschene K, Cu00e9leste C, Boerboom D, Theoret CL. Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1.. J Dermatol Sci 2012 Jan;65(1):12-8.
- Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Glowacki J. Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts.. Aging Cell 2008 Jun;7(3):335-43.
- Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion.. Stem Cells 2004;22(5):675-82.
- Scharstuhl A, Schewe B, Benz K, Gaissmaier C, Bu00fchring HJ, Stoop R. Chondrogenic potential of human adult mesenchymal stem cells is independent of age or osteoarthritis etiology.. Stem Cells 2007 Dec;25(12):3244-51.
- Fossett E, Khan WS, Longo UG, Smitham PJ. Effect of age and gender on cell proliferation and cell surface characterization of synovial fat pad derived mesenchymal stem cells.. J Orthop Res 2012 Jul;30(7):1013-8.
- Mathieu J, Zhou W, Xing Y, Sperber H, Ferreccio A, Agoston Z, Kuppusamy KT, Moon RT, Ruohola-Baker H. Hypoxia-inducible factors have distinct and stage-specific roles during reprogramming of human cells to pluripotency.. Cell Stem Cell 2014 May 1;14(5):592-605.
- Ezashi T, Das P, Roberts RM. Low O2 tensions and the prevention of differentiation of hES cells.. Proc Natl Acad Sci U S A 2005 Mar 29;102(13):4783-8.
- Forristal CE, Wright KL, Hanley NA, Oreffo RO, Houghton FD. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.. Reproduction 2010 Jan;139(1):85-97.
- Zhou W, Choi M, Margineantu D, Margaretha L, Hesson J, Cavanaugh C, Blau CA, Horwitz MS, Hockenbery D, Ware C, Ruohola-Baker H. HIF1u03b1 induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition.. EMBO J 2012 May 2;31(9):2103-16.
- Carbognin E, Betto RM, Soriano ME, Smith AG, Martello G. Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.. EMBO J 2016 Mar 15;35(6):618-34.
- Wu J, Yamauchi T, Izpisua Belmonte JC. An overview of mammalian pluripotency.. Development 2016 May 15;143(10):1644-8.
- Tristan C, Shahani N, Sedlak TW, Sawa A. The diverse functions of GAPDH: views from different subcellular compartments.. Cell Signal 2011 Feb;23(2):317-23.
- Ramzan R, Weber P, Linne U, Vogt S. GAPDH: the missing link between glycolysis and mitochondrial oxidative phosphorylation?. Biochem Soc Trans 2013 Oct;41(5):1294-7.
- Nicholls C, Li H, Liu JP. GAPDH: a common enzyme with uncommon functions.. Clin Exp Pharmacol Physiol 2012 Aug;39(8):674-9.
- Ralser M, Wamelink MM, Latkolik S, Jansen EE, Lehrach H, Jakobs C. Metabolic reconfiguration precedes transcriptional regulation in the antioxidant response.. Nat Biotechnol 2009 Jul;27(7):604-5.
- Son MY, Choi H, Han YM, Cho YS. Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency.. Stem Cells 2013 Nov;31(11):2374-87.
- Folmes CD, Nelson TJ, Martinez-Fernandez A, Arrell DK, Lindor JZ, Dzeja PP, Ikeda Y, Perez-Terzic C, Terzic A. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming.. Cell Metab 2011 Aug 3;14(2):264-71.
- Li XB, Gu JD, Zhou QH. Review of aerobic glycolysis and its key enzymes - new targets for lung cancer therapy.. Thorac Cancer 2015 Jan;6(1):17-24.
- Christensen DR, Calder PC, Houghton FD. GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells.. Sci Rep 2015 Dec 7;5:17500.
- Zhang J, Nuebel E, Daley GQ, Koehler CM, Teitell MA. Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal.. Cell Stem Cell 2012 Nov 2;11(5):589-95.
- Folmes CD, Arrell DK, Zlatkovic-Lindor J, Martinez-Fernandez A, Perez-Terzic C, Nelson TJ, Terzic A. Metabolome and metaboproteome remodeling in nuclear reprogramming.. Cell Cycle 2013 Aug 1;12(15):2355-65.
- Romesser PB, Romanyshyn JC, Schupak KD, Setton J, Riaz N, Wolden SL, Gelblum DY, Sherman EJ, Kraus D, Lee NY. Percutaneous endoscopic gastrostomy in oropharyngeal cancer patients treated with intensity-modulated radiotherapy with concurrent chemotherapy.. Cancer 2012 Dec 15;118(24):6072-8.
- Chen CT, Hsu SH, Wei YH. Mitochondrial bioenergetic function and metabolic plasticity in stem cell differentiation and cellular reprogramming.. Biochim Biophys Acta 2012 May;1820(5):571-6.
- Prieto J, Leu00f3n M, Ponsoda X, Garcu00eda-Garcu00eda F, Bort R, Serna E, Barneo-Muu00f1oz M, Palau F, Dopazo J, Lu00f3pez-Garcu00eda C, Torres J. Dysfunctional mitochondrial fission impairs cell reprogramming.. Cell Cycle 2016 Dec;15(23):3240-3250.
- Son MJ, Kwon Y, Son MY, Seol B, Choi HS, Ryu SW, Choi C, Cho YS. Mitofusins deficiency elicits mitochondrial metabolic reprogramming to pluripotency.. Cell Death Differ 2015 Dec;22(12):1957-69.
- Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.. Proc Natl Acad Sci U S A 1995 Jun 6;92(12):5510-4.
- Pouyssu00e9gur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression.. Nature 2006 May 25;441(7092):437-43.
- Pimton P, Lecht S, Stabler CT, Johannes G, Schulman ES, Lelkes PI. Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.. Stem Cells Dev 2015 Mar 1;24(5):663-76.
Citations
This article has been cited 4 times.- Barrachina L, Arshaghi TE, O'Brien A, Ivanovska A, Barry F. Induced pluripotent stem cells in companion animals: how can we move the field forward?. Front Vet Sci 2023;10:1176772.
- Recchia K, Pessu00f4a LVF, Pieri NCG, Pires PRL, Bressan FF. Influence of Cell Type in In Vitro Induced Reprogramming in Cattle.. Life (Basel) 2022 Jul 28;12(8).
- Recchia K, Machado LS, Botigelli RC, Pieri NCG, Barbosa G, de Castro RVG, Marques MG, Pessu00f4a LVF, Fantinato Neto P, Meirelles FV, de Souza AF, Martins SMMK, Bressan FF. In vitro induced pluripotency from urine-derived cells in porcine.. World J Stem Cells 2022 Mar 26;14(3):231-244.
- Bessi BW, Botigelli RC, Pieri NCG, Machado LS, Cruz JB, de Moraes P, de Souza AF, Recchia K, Barbosa G, de Castro RVG, Nogueira MFG, Bressan FF. Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation.. Cells 2021 Jun 17;10(6).