Cytotechnology

, Volume 70, Issue 1, pp 397–413 | Cite as

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

  • Monire Amerion
  • Mojtaba Rezazadeh Valojerdi
  • Saeid Abroun
  • Mehdi Totonchi
Original Article
  • 124 Downloads

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.

Keywords

Adipose derived stem cells Endothelial progenitor like cells Endothelial cells differentiation 

Notes

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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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Monire Amerion
    • 1
  • Mojtaba Rezazadeh Valojerdi
    • 1
    • 2
  • Saeid Abroun
    • 3
  • Mehdi Totonchi
    • 4
  1. 1.Department of Anatomy, Faculty of Medical ScienceTarbiat Modares UniversityTehranIslamic Republic of Iran
  2. 2.Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive BiomedicineACECRTehranIslamic Republic of Iran
  3. 3.Department of Hematology, Faculty of Medical SciencesTarbiat Modares UniversityTehranIslamic Republic of Iran
  4. 4.Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive BiomedicineACECRTehranIslamic Republic of Iran

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