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Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation

Published online by Cambridge University Press:  29 April 2022

Fatemeh Choupani ,
Vahideh Assadollahi ,
Zakaria Vahabzadeh ,
Erfan Daneshi ,
Morteza Abouzaripour ,
Farzad Soleimani ,
Saman Bahrami  and
Fardin Fathi Open the ORCID record for Fardin Fathi [Opens in a new window]
Fatemeh Choupani
Affiliation:
Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Vahideh Assadollahi
Affiliation:
Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Zakaria Vahabzadeh
Affiliation:
Department of Clinical Biochemistry, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran
Erfan Daneshi
Affiliation:
Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran
Morteza Abouzaripour
Affiliation:
Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran
Farzad Soleimani
Affiliation:
Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Saman Bahrami
Affiliation:
Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Fardin Fathi*
Affiliation:
Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
*
Author for correspondence: Fardin Fathi. Kurdistan University of Medical Sciences, Pasdaran St, Sanandaj, Iran 6617713446. Tel: +98 8733235445. Fax: +98 8733233600. E-mail: [email protected]
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Summary

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

Keywords

Fibroblast growth factor-2 (Fgf2)Inbred miceLeukaemia inhibitory factor (Lif)Mouse embryonic fibroblasts (MEFs)Outbred strain
Type
Research Article
Information
Zygote , Volume 30 , Issue 4 , August 2022 , pp. 550 - 560
Copyright
© The Author(s), 2022. Published by Cambridge University Press

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References

Abavisani, A, McKinnon, A, Tecirlioglu, RT, Trounson, AO and Guo, J (2010). Maintenance of horse embryonic stem cells in different conditions. Iran J Vet Res 11, 239–48.Google Scholar
Assadollahi, V, Fathi, F, Abdi, M, Khadem Erfan, MB, Soleimani, F and Banafshi, O (2019a). Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells. J Cell Biochem 120, 3716–26.CrossRefGoogle ScholarPubMed
Assadollahi, V, Hassanzadeh, K, Abdi, M, Alasvand, M, Nasseri, S and Fathi, F (2019b). Effect of embryo cryopreservation on derivation efficiency, pluripotency, and differentiation capacity of mouse embryonic stem cells. J Cell Physiol 234, 21962–72.CrossRefGoogle ScholarPubMed
Assadollahi, V, Mohammadi, E, Fathi, F, Hassanzadeh, K, Erfan, MBK, Soleimani, F, Banafshi, O, Yosefi, F and Allahvaisi, O (2019c). Effects of cigarette smoke condensate on proliferation and pluripotency gene expression in mouse embryonic stem cells. J Cell Biochem 120, 4071–80.CrossRefGoogle ScholarPubMed
Azizi, H, Conrad, S, Hinz, U, Asgari, B, Nanus, D, Peterziel, H, Hajizadeh Moghaddam, A, Baharvand, H and Skutella, T (2016). Derivation of pluripotent cells from mouse SSCs seems to be age dependent. Stem Cells Int 2016, 8216312.CrossRefGoogle ScholarPubMed
Azizi, H, Hamidabadi, HG and Skutella, T (2019). Differential proliferation effects after short-term cultivation of mouse spermatogonial stem cells on different feeder layers. Cell J 12, 186–93.Google Scholar
Barclay, C, Li, AW, Geldenhuys, L, Baguma-Nibasheka, M, Porter, GA, Veugelers, PJ, Murphy, PR and Casson, AG (2005). Basic fibroblast growth factor (FGF-2) overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. Clin Cancer Res 11, 7683–91.CrossRefGoogle ScholarPubMed
Beattie, GM, Lopez, AD, Bucay, N, Hinton, A, Firpo, MT, King, CC and Hayek, A (2005). Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells, 23, 489–95.CrossRefGoogle ScholarPubMed
Behringer, R, Gertsenstein, M, Nagy, KV and Nagy, A (2014). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press.Google Scholar
Brons, IGM, Smithers, LE, Trotter, MW, Rugg-Gunn, P, Sun, B, Chuva de Sousa Lopes, SMC, Howlett, SK, Clarkson, A, Ährlund-Richter, L, Pedersen, RA and Vallier, L (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–5.CrossRefGoogle ScholarPubMed
Chen, YF, Dong, Z, Jiang, L, Lai, D and Guo, L (2013). Mouse primed embryonic stem cells could be maintained and reprogrammed on human amnion epithelial cells. Stem Cell Dev 22, 320–9.CrossRefGoogle ScholarPubMed
Choi, KH, Lee, DK, Kim, SW, Woo, SH, Kim, DY and Lee, CK (2019). Chemically defined media can maintain pig pluripotency network in vitro . Stem Cell Rep 13, 221–34.CrossRefGoogle ScholarPubMed
Doetschman, T, Williams, P and Maeda, N (1988). Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev Biol 127, 224–7.CrossRefGoogle ScholarPubMed
Durkin, ME, Qian, X, Popescu, NC and Lowy, DR (2013). Isolation of mouse embryo fibroblasts. Bio Protoc 3, e908.CrossRefGoogle ScholarPubMed
Dutton, LC, Dudhia, J, Guest, DJ and Connolly, DJ (2019). Inducing pluripotency in the domestic cat (Felis catus). Stem Cells Dev 28, 1299–309.CrossRefGoogle Scholar
Dvorak, P, Dvorakova, D, Koskova, S, Vodinska, M, Najvirtova, M, Krekac, D and Hampl, A (2005). Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cell 23, 1200–11.CrossRefGoogle ScholarPubMed
Eiselleova, L, Peterkova, I, Neradil, J, Slaninova, I, Hampl, A and Dvorak, P (2008). Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol 52, 353–63.CrossRefGoogle ScholarPubMed
Evans, MJ and Kaufman, MH (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–6.CrossRefGoogle ScholarPubMed
Ezashi, T, Yuan, Y and Roberts, RM (2016). Pluripotent stem cells from domesticated mammals. Ann Rev Anim Biosci 4, 223–53.CrossRefGoogle ScholarPubMed
Fang, R, Liu, K, Zhao, Y, Li, H, Zhu, D, Du, Y, Xiang, C, Li, X, Liu, H, Miao, Z, Zhang, X, Shi, Y, Yang, W, Xu, J and Deng, H (2014). Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts. Cell Stem Cell 15, 488–97.CrossRefGoogle ScholarPubMed
Fuet, A and Pain, B (2017). Chicken induced pluripotent stem cells: establishment and characterization. Methods Mol Biol 1650, 211–28.CrossRefGoogle ScholarPubMed
Ginis, I, Luo, Y, Miura, T, Thies, S, Brandenberger, R, Gerecht-Nir, S, Amit, M, Hoke, A, Carpenter, MK, Itskovitz-Eldor, J and Rao, MS (2004). Differences between human and mouse embryonic stem cells. Dev Biol 269, 360–80.CrossRefGoogle ScholarPubMed
Gjørret, JO and Maddox-Hyttel, P (2005). Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures. Reprod Fertil Dev 17, 113–24.Google ScholarPubMed
Gómez, MC, Serrano, MA, Pope, CE, Jenkins, JA, Biancardi, MN, López, M, Dumas, C, Galiguis, J and Dresser, BL (2010). Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells. Theriogenology 74, 498–515.CrossRefGoogle ScholarPubMed
Hashimoto, M, Kondo, S, Sakurai, T, Etoh, Y, Shibai, H and Muramatsu, M (1990). Activin/EDF as an inhibitor of neural differentiation. Biochem Biophys Res Commun 173, 193–200.CrossRefGoogle ScholarPubMed
Honda, A (2013). Isolation and culture of rabbit embryonic stem cells. Methods Mol Biol 1074, 39–49.CrossRefGoogle ScholarPubMed
Honda, A, Hirose, M and Ogura, A (2009). Basic FGF and Activin/Nodal but not LIF signalling sustain undifferentiated status of rabbit embryonic stem cells. Exp Cell Res 315, 2033–42.CrossRefGoogle ScholarPubMed
Honda, A, Hirose, M, Hatori, M, Matoba, S, Miyoshi, H, Inoue, K and Ogura, A (2010). Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine. J Biol Chem 285, 31362–9.CrossRefGoogle ScholarPubMed
Hongisto, H, Mikhailova, A, Hiidenmaa, H, Ilmarinen, T and Skottman, H (2012). Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells. Stem Cell Discov 02, 176–86.CrossRefGoogle Scholar
Kanatsu-Shinohara, M, Ogonuki, N, Inoue, K, Miki, H, Ogura, A, Toyokuni, S and Shinohara, T (2003). Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69, 612–6.CrossRefGoogle ScholarPubMed
Kobayashi, M, Takada, T, Takahashi, K, Noda, Y and Torii, R (2008). BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells. Cloning Stem Cells 10, 495–502.CrossRefGoogle Scholar
Kubota, H, Avarbock, MR and Brinster, RL (2004). Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101, 16489–94.CrossRefGoogle ScholarPubMed
Lengner, CJ, Lepper, C, Van Wijnen, AJ, Stein, JL, Stein, GS and Lian, JB (2004). Primary mouse embryonic fibroblasts: A model of mesenchymal cartilage formation. J Cell Physiol 200, 327–33.CrossRefGoogle Scholar
Li, C, Yang, Y, Gu, J, Ma, Y and Jin, Y (2009). Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts. Cell Res 19, 173–86.CrossRefGoogle ScholarPubMed
Li, M, Zhang, D, Hou, Y, Jiao, L, Zheng, X and Wang, WH (2003). Isolation and culture of embryonic stem cells from porcine blastocysts. Mol Reprod Dev 65, 429–34.CrossRefGoogle ScholarPubMed
Li, M, Ma, W, Hou, Y, Sun, XF, Sun, QY and Wang, WH (2004). Improved isolation and culture of embryonic stem cells from Chinese miniature pig. J Reprod Dev 50, 237–44.CrossRefGoogle ScholarPubMed
Li, X, Zhou, SG, Imreh, MP, Ährlund-Richter, L and Allen, WR (2006). Horse embryonic stem cell lines from the proliferation of inner cell mass cells. Stem Cell Dev 15, 523–31.CrossRefGoogle ScholarPubMed
Lim, JWE and Bodnar, A (2002). Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2, 1187–203.3.0.CO;2-T>CrossRefGoogle ScholarPubMed
Liu, J, Balehosur, D, Murray, B, Kelly, JM, Sumer, H and Verma, PJ (2012). Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology 77, 338–46.e1.CrossRefGoogle ScholarPubMed
Liu, L, Peng, Z, Xu, Z, Huang, H and Wei, X (2018). Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells. J Mol Histol 49, 17–26.CrossRefGoogle ScholarPubMed
Llames, S, García-Pérez, E, Meana, Á, Larcher, F and del Río, M (2015). Feeder-layer cell actions and applications. Tissue Eng. Part B Rev 21, 345–53.CrossRefGoogle ScholarPubMed
Martin, GR (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634–8.CrossRefGoogle ScholarPubMed
Niwa, H (2001). Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct 26, 137–48.CrossRefGoogle ScholarPubMed
Ohtsuka, S, Nakai-Futatsugi, Y and Niwa, H (2015). LIF signal in mouse embryonic stem cells. JAKSTAT 4, e1086520.Google ScholarPubMed
Pain, B, Chenevier, P and Samarut, J (1999). Chicken embryonic stem cells and transgenic strategies. Cell Tissue Organ 165, 212–9.CrossRefGoogle ScholarPubMed
Park, TS and Han, JY (2000). Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 56, 475–82.3.0.CO;2-M>CrossRefGoogle ScholarPubMed
Pau, KY and Wolf, DP (2004). Derivation and characterization of monkey embryonic stem cells. Reprod Biol Endocrinol 2, 41.CrossRefGoogle ScholarPubMed
Pei, H, Fu, HY, Hirai, H, Cho, DS, O’Brien, TD, Dutton, J, Verfaillie, CM and Hu, WS (2017). Generation of induced pluripotent stem cells from Chinese hamster embryonic fibroblasts. Stem Cell Res 21, 132–6.CrossRefGoogle ScholarPubMed
Pekkanen-Mattila, M, Ojala, M, Kerkelä, E, Rajala, K, Skottman, H and Aalto-Setälä, K (2012). The effect of human and mouse fibroblast feeder cells on cardiac differentiation of human pluripotent stem cells. Stem Cell Int 2012, 875059.Google ScholarPubMed
Rajarajan, K, Engels, MC and Wu, SM (2012). Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells. Curr Protoc Mol Biol, Chapter Unit–23.15, doi: 10.1002/0471142727.mb2315s97.CrossRefGoogle ScholarPubMed
Richards, M, Tan, S, Fong, CY, Biswas, A, Chan, WK and Bongso, A (2003). Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cell 21, 546–56.CrossRefGoogle ScholarPubMed
Saha, S, Ji, L, De Pablo, JJ and Palecek, SP (2008). TGF-β/Activin/Nodal pathway in inhibition of human embryonic stem cell differentiation by mechanical strain. Biophys J 94, 4123–33.CrossRefGoogle Scholar
Schmitt, RM, Bruyns, E and Snodgrass, HR (1991). Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor gene expression. Genes Dev 5, 728–40.CrossRefGoogle ScholarPubMed
Schnabel, LV, Abratte, CM, Schimenti, JC, Southard, TL and Fortier, LA (2012). Genetic background affects induced pluripotent stem cell generation. Stem Cell Res Ther 3, 30.CrossRefGoogle ScholarPubMed
Schuldiner, M, Yanuka, O, Itskovitz-Eldor, J, Melton, DA and Benvenisty, N (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97, 11307–12.CrossRefGoogle ScholarPubMed
Sharma, R, George, A, Kamble, NM, Singh, KP, Chauhan, MS, Singla, SK, Manik, RS and Palta, P (2011). Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells. Cell Reprogram 13, 539–49.CrossRefGoogle ScholarPubMed
Shimozawa, N, Ono, R, Shimada, M, Shibata, H, Takahashi, I, Inada, H, Takada, T, Nosaka, T and Yasutomi, Y (2013). Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species. Differentiation 85, 131–9.CrossRefGoogle ScholarPubMed
Soleimani, F, Babaei, E, Feizi, MA and Fathi, F (2020). CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes. J Cell Physiol 235, 3994–4000.CrossRefGoogle Scholar
Sulzbacher, S, Schroeder, IS, Truong, TT and Wobus, AM (2009). Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors—The influence of differentiation factors and culture conditions. Stem Cell Rev Rep 5, 159–73.CrossRefGoogle ScholarPubMed
Sun, L, Bradford, CS, Ghosh, C, Collodi, P and Barnes, DW (1995). ES-like cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotechnol 4, 193–9.Google ScholarPubMed
Takahashi, K and Yamanaka, S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–76.CrossRefGoogle ScholarPubMed
Tao, T, Yang, S, Zhang, J, Wang, S, Tang, X and Ji, W (2008). Differentiation of rabbit embryonic stem cells into trophoblast stem cell. Cell Res 18 (Suppl. 1), S149.CrossRefGoogle Scholar
Teotia, P, Sharma, S, Airan, B and Mohanty, S (2016). Feeder and basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders. Indian J Med Res 144, 838–51.Google ScholarPubMed
Tesar, PJ, Chenoweth, JG, Brook, FA, Davies, TJ, Evans, EP, Mack, DL, Gardner, RL and McKay, RD (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–9.CrossRefGoogle ScholarPubMed
Thomson, JA, Itskovitz-Eldor, J, Shapiro, SS, Waknitz, MA, Swiergiel, JJ, Marshall, VS and Jones, JM (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–7.CrossRefGoogle ScholarPubMed
Tilgner, K, Atkinson, SP, Golebiewska, A, Stojković, M, Lako, M and Armstrong, L (2008). Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 26, 3075–85.CrossRefGoogle ScholarPubMed
Tomizawa, M, Shinozaki, F, Sugiyama, T, Yamamoto, S, Sueishi, M and Yoshida, T (2011). Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells. Exp Ther Med 2, 405–8.CrossRefGoogle ScholarPubMed
Ueda, S, Kawamata, M, Teratani, T, Shimizu, T, Tamai, Y, Ogawa, H, Hayashi, K, Tsuda, H and Ochiya, T (2008). Establishment of rat embryonic stem cells and making of chimera rats. PLoS One 3, e2800.CrossRefGoogle ScholarPubMed
Vogel, C and Marcotte, EM (2012). Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 13, 227–32.CrossRefGoogle ScholarPubMed
Xiao, L, Yuan, X and Sharkis, SJ (2006). Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells 24, 1476–86.CrossRefGoogle ScholarPubMed
Xie, CQ, Lin, G, Luo, KL, Luo, SW and Lu, GX (2004). Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive. Biochem Biophys Res Commun 315, 581–8.CrossRefGoogle ScholarPubMed
Xu, RH, Chen, X, Li, DS, Li, R, Addicks, GC, Glennon, C, Zwaka, TP and Thomson, JA (2002). BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20, 1261–4.CrossRefGoogle ScholarPubMed
Xu, RH, Peck, RM, Li, DS, Feng, X, Ludwig, T and Thomson, JA (2005). Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Method 2, 185–90.CrossRefGoogle ScholarPubMed
Yang, F, Wang, N, Wang, Y, Yu, T and Wang, H (2017). Activin-SMAD signaling is required for maintenance of porcine iPS cell self-renewal through upregulation of NANOG and OCT4 expression. J Cell Physiol 232, 2253–62.CrossRefGoogle ScholarPubMed
Yang, H, Qiu, Y, Zeng, X, Ding, Y, Zeng, J, Lu, K and Li, D (2016). Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells. Exp Ther Med 11, 2321–8.CrossRefGoogle ScholarPubMed
Zarei Fard, NZ, Talaei-Khozani, T, Bahmanpour, S and Esmaeilpour, T (2015). Comparison of cell viability and embryoid body size of two embryonic stem cell lines after different exposure times to bone morphogenetic protein 4. Iran J Med Sci 40, 110–7.Google ScholarPubMed
Zhang, P, Li, J, Tan, Z, Wang, C, Liu, T, Chen, L, Yong, J, Jiang, W, Sun, X, Du, L, Ding, M and Deng, H (2008). Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood 111, 1933–41.CrossRefGoogle ScholarPubMed
Zou, Q, Wu, M, Zhong, L, Fan, Z, Zhang, B, Chen, Q and Ma, F (2016). Development of a xeno-free feeder-layer system from human umbilical cord mesenchymal stem cells for prolonged expansion of human induced pluripotent stem cells in culture. PLoS One 11, e0149023.CrossRefGoogle ScholarPubMed