In this study, CD34+/CD31− progenitor cells were isolated from the stromal vascular fraction (SVF) of adipose tissue using magnetic activated cell sorting. The endothelial differentiation capability of these cells in vitro was evaluated by culturing them in vascular endothelial growth factor (VEGF)–induced medium for 14 days. Viability, proliferation, differentiation, and tube formation of these cells were evaluated. Cell viability study revealed that both undifferentiated and endothelial-differentiated cells remained healthy for 14 days. However, the proliferation rate was higher in undifferentiated cells compared to endothelial-differentiated ones. Upregulation of endothelial characteristic genes (von Willebrand factor (vWF) and VE-cadherin) was observed in 2D culture. However, PECAM (CD31) was only found to be upregulated after the cells had formed tube-like structures in 3D Matrigel culture. These results indicate that adipose-derived CD34+/CD31− cells when cultured in VEGF-induced medium, are capable differentiation into endothelial-like lineages. Tube formation of the cells started 3 h after seeding the cells on Matrigel and formed more stable and connected network 24 h post-seeding in presence of VEGF.
In this work, endothelial differentiation capability of CD34+/CD31− cells isolated from adipose tissue was evaluated. The results showed that these cells can be successfully differentiated into endothelial cells in vitro in the presence of VEGF. Vascularization is crucial for the survival of the cells and formation of new tissues in engineered tissue constructs. Therefore, finding a proper source of progenitor cells capable of endothelial differentiation is of substantial importance in tissue engineering for the vascularization of large-volume complex tissues. CD34+/CD31− cells can be easily harvested from adipose tissues in abundance unlike other mesenchymal stem cells such as bone marrow stem cells, which makes them advantageous for vascularization in tissue engineering.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Wang N, Wang S-J, Zhang R, Zhang T-C, Zhang C-L, Mao L-B, et al. Vascular endothelial growth factor stimulates endothelial differentiation from mesenchymal stem cells via rho/myocardin-related transcription factor-a signaling pathway. Int J Biochem Cell Biol. 2013;45(7):1447–56.
Rouwkema J, Khademhosseini A. Vascularization and angiogenesis in tissue engineering: beyond creating static networks. Trends Biotechnol. 2016;34(9):733–45.
Asahara T, Murohara T, Sullivan A, Silver M, van der Rien Z, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–7.
Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H, et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation. 2006;113(10):1311–25.
Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, et al. CD146 coordinates brain endothelial cell–pericyte communication for blood–brain barrier development. Proc Natl Acad Sci U S A. 2017;114(36):E7622–E31.
Frese L, Dijkman PE, Hoerstrup SP. Adipose tissue-derived stem cells in regenerative medicine. Transfus Med Hemother. 2016;43(4):268–74.
Lindner U, Kramer J, Rohwedel J, Schlenke P. Mesenchymal stem or stromal cells: toward a better understanding of their biology? Transfus Med Hemother. 2010;37(2):75–83.
Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cells Int. 2012;2012:812693.
Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249–60.
Coralie S, Alexandra M, Marie M, Sandra DB, Rudi B, Anne B. Chemotaxis and differentiation of human adipose tissue CD34+/CD31−progenitor cells: role of stromal derived Factor-1 released by adipose tissue capillary endothelial cells. Stem Cells. 2007;25(9):2269–76.
Hertweck J, Ritz U, Götz H, Schottel PC, Rommens PM, Hofmann A. CD34+ cells seeded in collagen scaffolds promote bone formation in a mouse calvarial defect model. J Biomed Mater Res B Appl Biomater. 2018;106(4):1505–16.
Miranville A, Heeschen C, Sengenes C, Curat CA, Busse R, Bouloumie A. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation. 2004;110(3):349–55.
Yang JJ, Ii M, Kamei N, Alev C, Kwon SM, Kawamoto A, et al. CD34(+) cells represent highly functional endothelial progenitor cells in murine bone marrow. PLoS One. 2011;6(5):e20219.
Liu Q, Cen L, Yin S, Chen L, Liu G, Chang J, et al. A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and β-TCP ceramics. Biomaterials. 2008;29(36):4792–9.
Zanetti AS, McCandless GT, Chan JY, Gimble JM, Hayes DJ. Characterization of novel akermanite:poly-ϵ-caprolactone scaffolds for human adipose-derived stem cells bone tissue engineering. J Tissue Eng Regen Med. 2015;9(4):389–404.
Lian X, Bao X, Al-Ahmad A, Liu J, Wu Y, Dong W, et al. Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Stem Cell Rep. 2014;3(5):804–16.
Behr B, Tang C, Germann G, Longaker MT, Quarto N. Locally applied vascular endothelial growth factor a increases the osteogenic healing capacity of human adipose-derived stem cells by promoting osteogenic and endothelial differentiation. Stem Cells. 2011;29(2):286–96.
Zonari A, Novikoff S, Electo NRP, Breyner NM, Gomes DA, Martins A, et al. Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh. PLoS One. 2012;7(4):e354e22.
Unger RE, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials. 2007;28(27):3965–76.
Fang TD, Salim A, Xia W, Nacamuli RP, Guccione S, Song HM, et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 2005;20(7):1114–24.
Peters EB. Endothelial progenitor cells for the vascularization of engineered tissues. Tissue Eng B Rev. 2018;24(1):1–24.
Portalska KK, Leferink AM, Groen N, Fernandes HAM, Moroni L, van Blitterswijk C, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One. 2012;7(10):1–16.
Zhang P, Moudgill N, Hager E, Tarola N, DiMatteo C, McIlhenny S, et al. Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease. Stem Cells Dev. 2011;20(6):977–88.
Dao TT-T, Vu NB, Phi LT, Ha Thi-Ngan L, Phan NK, Ta VT, et al. Human adipose-derived mesenchymal stem cell could participate in angiogenesis in a mouse model of acute hindlimb ischemia. Biomed Res Ther. 2016;3(8):770–9.
Suga H, Matsumoto D, Eto H, Inoue K, Aoi N, Kato H, et al. Functional implications of CD34 expression in human adipose–derived stem/progenitor cells. Stem Cells Dev. 2009;18(8):1201–10.
Oswald J, Boxberger S, Jørgensen B, Feldmann S, Ehninger G, Bornhäuser M, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells. 2004;22(3):377–84.
Wang C, Li Y, Yang M, Zou Y, Liu H, Liang Z, et al. Efficient differentiation of bone marrow mesenchymal stem cells into endothelial cells in vitro. Eur J Vasc Endovasc Surg. 2018;55(2):257–65.
Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A. 2002;99(7):4391–6.
van den Heuvel S. Coordinating cell proliferation and differentiation: antagonism between cell cycle regulators and cell type-specific gene expression AU – Ruijtenberg, Suzan. Cell Cycle. 2016;15(2):196–212.
Cooper GM. The cell - a molecular approach 2nd edition. Sunderland: Sinauer Associates; 2000.
Zhang G, Zhang F, Zhou J, Fan Q, Zheng Z, Liu X, et al. Arterial–venous endothelial cell fate is related to vascular endothelial growth factor and notch status during human bone mesenchymal stem cell differentiation. FEBS Lett. 2008;582(19):2957–64.
Chen M-Y, Lie P-C, Li Z-L, Wei X. Endothelial differentiation of Wharton’s jelly–derived mesenchymal stem cells in comparison with bone marrow–derived mesenchymal stem cells. Exp Hematol. 2009;37(5):629–40.
Cao G, O’Brien CD, Zhou Z, Sanders SM, Greenbaum JN, Makrigiannakis A, et al. Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration. Am J Physiol Cell Physiol. 2002;282(5):1181–90.
Matsumura T, Wolff K, Petzelbauer P. Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin. J Immunol. 1997;158(7):3408–16.
Kleinman HK, Arnaoutova I. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 2010;5(4):628–35.
Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol. 2005;15(5):378–86.
Arnaoutova I, George J, Kleinman HK, Benton G. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 2009;12(3):267–74.
Oliva-Olivera W, Coín-Aragüez L, Lhamyani S, Salas J, Gentile A-M, Romero-Zerbo S-Y, et al. Differences in the neovascular potential of thymus versus subcutaneous adipose-derived stem cells from patients with myocardial ischaemia. J Tissue Eng Regen Med. 2018;12(3):e1772–e84.
Zongning M, Jun J, Lei C, Jianzhong Z, Wei H, Jidong Z, et al. Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int. 2006;30(9):681–7.
Traktuev DO, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, et al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008;102(1):77–85.
Boquest AC, Shahdadfar A, Frønsdal K, Sigurjonsson O, Tunheim SH, Collas P, et al. Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell. 2005;16(3):1131–41.
Oñate B, Vilahur G, Ferrer-Lorente R, Ybarra J, Díez-Caballero A, Ballesta-López C, et al. The subcutaneous adipose tissue reservoir of functionally active stem cells is reduced in obese patients. FASEB J. 2012;26(10):4327–36.
Oliva-Olivera W, Lhamyani S, Coín-Aragüez L, Castellano-Castillo D, Alcaide-Torres J, Yubero-Serrano EM, et al. Neovascular deterioration, impaired NADPH oxidase and inflammatory cytokine expression in adipose-derived multipotent cells from subjects with metabolic syndrome. Metabolism. 2017;71:132–43.
Oliva-Olivera W, Moreno-Indias I, Coín-Aragüez L, Lhamyani S, Alcaide Torres J, Fernández-Veledo S, et al. Different response to hypoxia of adipose-derived multipotent cells from obese subjects with and without metabolic syndrome. PloS one. 2017;12(11):e0188324-e.
Efimenko A, Dzhoyashvili N, Kalinina N, Kochegura T, Akchurin R, Tkachuk V, et al. Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential. Stem Cells Transl Med. 2014;3(1):32–41.
Research reported in this article was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award number (RDE024790A).
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Forghani, A., Koduru, S.V., Chen, C. et al. Differentiation of Adipose Tissue–Derived CD34+/CD31− Cells into Endothelial Cells In Vitro. Regen. Eng. Transl. Med. 6, 101–110 (2020). https://doi.org/10.1007/s40883-019-00093-7
- Stromal vascular fraction
- CD34+ cells
- Endothelial differentiation
- Adipose tissue