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Long term culture and differentiation of endothelial progenitor like cells from rat adipose derived stem cells

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Abstract

The procedure of obtaining qualified endothelial progenitor cells (EPCs) is still unclear and there has been some controversy on their biological properties and time of emergence. In this study, we used long-term culture of Adipose Derived Stem Cells (ADSCs) in an endothelial induction medium to obtain endothelial progenitor-like cells, and investigated the features of a few surface markers and the physiologic functions of the cells produced. In order to achieve our aim, rat ADSCs were isolated and cultured in an endothelial basal medium (EBM2), supplemented with an endothelial growth medium (EGM2). The cells were cultured 1 week for short-time, 2 weeks for mid-time, and 3 weeks for long-time cultures. Morphological changes were monitored by phase contrast and electron microscopy. The expressions of a few endothelial progenitor cells markers were analyzed by real-time RT-PCR. Low-density lipoprotein uptake and lectin binding assay were also performed for functional characterization. After induction, ADSCs showed changes in morphology from spindle-shaped in the first week to cobblestone-shaped during the next 2 weeks. Then, endothelial cell phenotype was defined by the presence of Weibel–Palade bodies in the cytoplasm and tube formation, without the use of Matrigel in the third week. In keeping with gene expression analysis, VEGFR-2 showed significant expression during early stages of endothelial differentiation for up to 3 weeks. A significantly increased expression of Tie2 was observed on day 21. Likewise, VE-Cadherin, CD34, CD133, WVF and CD31 were not significantly expressed within the same period of time. Endothelial differentiated cells also showed little LDL uptake and little to no lectin binding during the first 2 weeks of induction. However, high LDL uptake and lectin binding were observed in the third week. It appears that long term culture of ADSCs in EGM2 leads to significantly increased expression of some endothelial progenitor cells markers, strong DiI-ac-LDL uptake, lectin binding and tube-like structure formation in endothelial differentiated cells. Therefore, selection of an appropriate culture time and culture medium is crucial for establishing an efficient route to obtain sufficient numbers of EPCs with optimized quantity and quality.

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References

  • Alphonse RS, Vadivel A, Zhong S, McConaghy S, Ohls R, Yoder MC, Thebaud B (2015) The isolation and culture of endothelial colony-forming cells from human and rat lungs. Nat Protoc 10:1697–1708. doi:10.1038/nprot.2015.107

    Article  CAS  Google Scholar 

  • Arnaoutova I, George J, Kleinman HK, Benton G (2009) The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis 12:267–274. doi:10.1007/s10456-009-9146-4

    Article  Google Scholar 

  • Asahara T et al (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967

    Article  CAS  Google Scholar 

  • Asahara T et al (1999) VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18:3964–3972. doi:10.1093/emboj/18.14.3964

    Article  CAS  Google Scholar 

  • Bellik L, Ledda F, Parenti A (2005) Morphological and phenotypical characterization of human endothelial progenitor cells in an early stage of differentiation. FEBS Lett 579:2731–2736. doi:10.1016/j.febslet.2005.04.003

    Article  CAS  Google Scholar 

  • Benavides OM, Petsche JJ, Moise KJ Jr, Johnson A, Jacot JG (2012) Evaluation of endothelial cells differentiated from amniotic fluid-derived stem cells. Tissue Eng Part A 18:1123–1131. doi:10.1089/ten.TEA.2011.0392

    Article  CAS  Google Scholar 

  • Cao Y, Sun Z, Liao L, Meng Y, Han Q, Zhao RC (2005) Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun 332:370–379. doi:10.1016/j.bbrc.2005.04.135

    Article  CAS  Google Scholar 

  • Cheng CC et al (2013) Distinct angiogenesis roles and surface markers of early and late endothelial progenitor cells revealed by functional group analyses. BMC Genom 14:182. doi:10.1186/1471-2164-14-182

    Article  CAS  Google Scholar 

  • Eggermann J et al (2003) Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood. Cardiovasc Res 58:478–486

    Article  CAS  Google Scholar 

  • Feng B, Liu YL, Feng K, Gong R, Chen H (2005) Evaluation of endothelial cells differentiated from mesenchymal stem cells of human bone marrow with Tie-2 monoclonal antibody by immunohistochemistry in vitro. Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chin J Appl Physiol 21:340–343

    Google Scholar 

  • Fischer LJ et al (2009) Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force. J Surg Res 152:157–166. doi:10.1016/j.jss.2008.06.029

    Article  CAS  Google Scholar 

  • George AL, Bangalore-Prakash P, Rajoria S, Suriano R, Shanmugam A, Mittelman A, Tiwari RK (2011) Endothelial progenitor cell biology in disease and tissue regeneration. J Hematol Oncol 4:24. doi:10.1186/1756-8722-4-24

    Article  Google Scholar 

  • Ghadge SK, Muhlstedt S, Ozcelik C, Bader M (2011) SDF-1alpha as a therapeutic stem cell homing factor in myocardial infarction. Pharmacol Ther 129:97–108. doi:10.1016/j.pharmthera.2010.09.011

    Article  CAS  Google Scholar 

  • Gregory CA, Gunn WG, Peister A, Prockop DJ (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 329:77–84. doi:10.1016/j.ab.2004.02.002

    Article  CAS  Google Scholar 

  • Guan L et al (2006) In vitro differentiation of human adipose-derived mesenchymal stem cells into endothelial-like cells. Chin Sci Bull 51:1863–1868

    Article  CAS  Google Scholar 

  • Guan XM et al (2013) Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media. Mol Med Rep 8:1722–1728. doi:10.3892/mmr.2013.1718

    Article  CAS  Google Scholar 

  • Hur J et al (2004) Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 24:288–293. doi:10.1161/01.ATV.0000114236.77009.06

    Article  CAS  Google Scholar 

  • Igreja C et al (2008) Detailed molecular characterization of cord blood-derived endothelial progenitors. Exp Hematol 36:193–203. doi:10.1016/j.exphem.2007.09.001

    Article  CAS  Google Scholar 

  • Ingram DA et al (2004) Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104:2752–2760. doi:10.1182/blood-2004-04-1396

    Article  CAS  Google Scholar 

  • Janeczek Portalska K, Leferink A, Groen N, Fernandes H, Moroni L, van Blitterswijk C, de Boer J (2012) Endothelial differentiation of mesenchymal stromal cells. PLoS ONE 7:e46842. doi:10.1371/journal.pone.0046842

    Article  Google Scholar 

  • Kang BJ, Lee SH, Kweon OK, Cho JY (2014) Differentiation of canine adipose tissue-derived mesenchymal stem cells towards endothelial progenitor cells. Am J Vet Res 75:685–691. doi:10.2460/ajvr.75.7.685

    Article  CAS  Google Scholar 

  • Kawamoto A, Losordo DW (2008) Endothelial progenitor cells for cardiovascular regeneration. Trends Cardiovasc Med 18:33–37. doi:10.1016/j.tcm.2007.11.004

    Article  CAS  Google Scholar 

  • Kellouche S et al (2007) Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation. Exp Cell Res 313:486–499. doi:10.1016/j.yexcr.2006.10.023

    Article  CAS  Google Scholar 

  • Kovacic JC, Boehm M (2009) Resident vascular progenitor cells: an emerging role for non-terminally differentiated vessel-resident cells in vascular biology. Stem Cell Res 2:2–15. doi:10.1016/j.scr.2008.05.005

    Article  Google Scholar 

  • Lee PS, Poh KK (2014) Endothelial progenitor cells in cardiovascular diseases. World J Stem Cells 6:355–366. doi:10.4252/wjsc.v6.i3.355

    Article  Google Scholar 

  • Liu JW et al (2007) Characterization of endothelial-like cells derived from human mesenchymal stem cells. J Thromb Haemost 5:826–834. doi:10.1111/j.1538-7836.2007.02381.x

    Article  CAS  Google Scholar 

  • Martinez-Estrada OM, Munoz-Santos Y, Julve J, Reina M, Vilaro S (2005) Human adipose tissue as a source of Flk-1+ cells: new method of differentiation and expansion. Cardiovasc Res 65:328–333. doi:10.1016/j.cardiores.2004.11.015

    Article  CAS  Google Scholar 

  • Ning H, Liu G, Lin G, Yang R, Lue TF, Lin CS (2009) Fibroblast growth factor 2 promotes endothelial differentiation of adipose tissue-derived stem cells. J Sex Med 6:967–979. doi:10.1111/j.1743-6109.2008.01172.x

    Article  CAS  Google Scholar 

  • Niyaz M, Gürpinar ÖA, Günaydin S, Onur MA (2012) Isolation, culturing and characterization of rat adipose tissue-derived mesenchymal stem cells: a simple technique. Turk J Biol 36:658–664

    Google Scholar 

  • Oshima-Sudo N, Li Q, Hoshino Y, Nakahama K-I, Kubota T, Morita I (2011) Optimized method for culturing outgrowth endothelial progenitor cells. Inflamm Regen 31:219–227

    Article  CAS  Google Scholar 

  • Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger G, Bornhauser M, Werner C (2004) Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22:377–384. doi:10.1634/stemcells.22-3-377

    Article  Google Scholar 

  • Ranjan AK, Kumar U, Hardikar AA, Poddar P, Nair PD, Hardikar AA (2009) Human blood vessel-derived endothelial progenitors for endothelialization of small diameter vascular prosthesis. PLoS ONE 4:e7718. doi:10.1371/journal.pone.0007718

    Article  Google Scholar 

  • Rehman J (2011) Chipping away at the surface of the endothelial progenitor cell (EPC) mystery. J Mol Med 89:943–945. doi:10.1007/s00109-011-0799-2

    Article  Google Scholar 

  • Reinisch A et al (2009) Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood 113:6716–6725. doi:10.1182/blood-2008-09-181362

    Article  CAS  Google Scholar 

  • Santos SC, Miguel C, Domingues I, Calado A, Zhu Z, Wu Y, Dias S (2007) VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing. Exp Cell Res 313:1561–1574. doi:10.1016/j.yexcr.2007.02.020

    Article  CAS  Google Scholar 

  • Smadja DM et al (2007) Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6). J Cell Mol Med 11:1149–1161. doi:10.1111/j.1582-4934.2007.00090.x

    Article  CAS  Google Scholar 

  • Song E, Lu CW, Fang LJ, Yang W (2010) Culture and identification of endothelial progenitor cells from human umbilical cord blood. Int J Ophthalmol 3:49–53. doi:10.3980/j.issn.2222-3959.2010.01.11

    CAS  Google Scholar 

  • Sun W, Zheng L, Han P, Kang YJ (2014) Isolation and characterization of endothelial progenitor cells from Rhesus monkeys. Regen Med Res 2:5. doi:10.1186/2050-490X-2-5

    Article  Google Scholar 

  • Tongers J, Losordo DW (2007) Frontiers in nephrology: the evolving therapeutic applications of endothelial progenitor cells. J Am Soc Nephrol 18:2843–2852. doi:10.1681/ASN.2007050597

    Article  Google Scholar 

  • van den Akker NM et al (2012) Vascular potency of Sus scrofa bone marrow-derived mesenchymal stem cells: a progenitor source of medial but not endothelial cells. Tissue Eng Part A 18:828–839. doi:10.1089/ten.TEA.2011.0284

    Article  Google Scholar 

  • Werling NJ, Thorpe R, Zhao Y (2013) A systematic approach to the establishment and characterization of endothelial progenitor cells for gene therapy. Hum Gene Ther Methods 24:171–184. doi:10.1089/hgtb.2012.146

    Article  CAS  Google Scholar 

  • Yang N et al (2011) The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions. Cytotechnology 63:217–226. doi:10.1007/s10616-010-9329-2

    Article  CAS  Google Scholar 

  • Yu G, Floyd ZE, Wu X, Hebert T, Halvorsen YD, Buehrer BM, Gimble JM (2011) Adipogenic differentiation of adipose-derived stem cells. Methods Mol Biol 702:193–200. doi:10.1007/978-1-61737-960-4_14

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by Grants from Tarbiat Modares University and Royan Institute (both located in Tehran, Iran). Auteurs would like to thank Dr. Vahid Pirhajati for his kind support in preparation of EM images.

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Authors and Affiliations

  1. Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, P.O.Box. 14115-331, Tehran, Islamic Republic of Iran

    Monire Amerion & Mojtaba Rezazadeh Valojerdi

  2. Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Islamic Republic of Iran

    Mojtaba Rezazadeh Valojerdi

  3. Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, P.O.Box.14115-331, Tehran, Islamic Republic of Iran

    Saeid Abroun

  4. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Islamic Republic of Iran

    Mehdi Totonchi

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  1. Monire Amerion
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Correspondence to Mojtaba Rezazadeh Valojerdi.

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Amerion, M., Valojerdi, M.R., Abroun, S. et al. Long term culture and differentiation of endothelial progenitor like cells from rat adipose derived stem cells. Cytotechnology 70, 397–413 (2018). https://doi.org/10.1007/s10616-017-0155-7

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