, Volume 71, Issue 1, pp 1–14 | Cite as

Bone marrow derived endothelial progenitor cells retain their phenotype and functions after a limited number of culture passages and cryopreservation

  • Xianghui GongEmail author
  • Bin Li
  • Yongxing Yang
  • Yan Huang
  • Yan Sun
  • Meili Liu
  • Xiaoling Jia
  • Yubo FanEmail author


A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and post-cryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications.


Endothelial progenitor cells Passage Cryopreservation Bone marrow 



This work was supported by the National Natural Science Foundation of China (Nos. 31771019, 11172031, 31470901). The national key research and development plan (No. 2016YFC1101101). International Joint Research Center of Aerospace Biotechnology and 344 Medical Engineering from Ministry of Science and Technology of China, 111 Project 345 (No. B13003).

Compliance with ethical standards

Conflict of interest

The authors have no additional conflict of interest to disclose.

Supplementary material

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  1. Asahara T et al (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967. CrossRefGoogle Scholar
  2. Bai CY, Hou L, Zhang M, Pu Y, Guan W, Ma Y (2012) Characterization of vascular endothelial progenitor cells from chicken bone marrow. BMC Vet Res 8:54. CrossRefGoogle Scholar
  3. Bogoslovsky T, Wang D, Maric D, Scattergood-Keepper L, Spatz M, Auh S, Hallenbeck J (2013) Cryopreservation and enumeration of human endothelial progenitor and endothelial cells for clinical trials. J Blood Disord Transfus 4:158Google Scholar
  4. Chabannes D et al (2007) A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. Blood 110:3691–3694. CrossRefGoogle Scholar
  5. Chen C, Zheng S, Zhang X, Dai P, Gao Y, Nan L, Zhang Y (2017) Transplantation of amniotic scaffold-seeded mesenchymal stem cells and/or endothelial progenitor cells from bone marrow to efficiently repair 3-cm circumferential urethral defect in model dogs. Tissue Eng Part A 24:47–56. CrossRefGoogle Scholar
  6. Chong MSK, Ng WK, Chan JKY (2016) Concise review: endothelial progenitor cells in regenerative medicine: applications and challenges. Stem Cell Transl Med 5:530–538. CrossRefGoogle Scholar
  7. Fadini GP, Coracina A, Baesso I, Agostini C, Tiengo A, Avogaro A, Vigili de Kreutzenberg S (2006) Peripheral blood CD34+KDR+ endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population. Stroke 37:2277–2282. CrossRefGoogle Scholar
  8. Garbuzova-Davis S, Haller E, Lin R, Borlongan CV (2017) Intravenously transplanted human bone marrow endothelial progenitor cells engraft within brain capillaries, preserve mitochondrial morphology, and display pinocytotic activity toward blood-brain barrier repair in ischemic stroke rats. Stem Cells 35:1246–1258. CrossRefGoogle Scholar
  9. Garolla A et al (2009) Reduced endothelial progenitor cell number and function in inflammatory bowel disease: a possible link to the pathogenesis. Am J Gastroenterol 104:2500–2507. CrossRefGoogle Scholar
  10. Hristov M, Erl W, Weber PC (2003) Endothelial progenitor cells mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol 23:1185–1189. CrossRefGoogle Scholar
  11. 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. CrossRefGoogle Scholar
  12. Javazon EH, Colter DC, Schwarz EJ, Prockop DJ (2001) Rat marrow sromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells 19:219–225. CrossRefGoogle Scholar
  13. Jianguo W, Tianhang L, Hong Z, Zhengmao L, Jianwei B, Xuchao X, Guoen F (2010) Optimization of culture conditions for endothelial progenitor cells from porcine bone marrow in vitro. Cell Prolif 43:418–426. CrossRefGoogle Scholar
  14. Kalka C et al (2000) Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 97:3422–3427. CrossRefGoogle Scholar
  15. Karlsson JOM (2002) Cryopreservation: freezing and vitrification. Science 296:655–656. CrossRefGoogle Scholar
  16. Kaushal S et al (2001) Functional small diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med 7:1035–1040. CrossRefGoogle Scholar
  17. Kim H, Kim S, Baek SH, Kwon SM (2016) Pivotal cytoprotective mediators and promising therapeutic strategies for endothelial progenitor cell-based cardiovascular regeneration. Stem Cells Int 2016:1–14. Google Scholar
  18. Kretlow JD et al (2008) Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 9:60. CrossRefGoogle Scholar
  19. Kuki S, Imanishi T, Kobayashi K, Matsuo Y, Obana M, Akasaka T (2006) Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen-activated protein kinase. Circ J 70:1076–1081. CrossRefGoogle Scholar
  20. Leeper NJ, Hunter AL, Cooke JP (2010) Stem cell therapy for vascular regeneration: adult, embryonic, and induced pluripotent stem cells. Circulation 122:517–526. CrossRefGoogle Scholar
  21. Lev EI et al (2006) Potential role of activated platelets in homing of human endothelial progenitor cells to subendothelial matrix. Thromb Haemost 96:498–504. CrossRefGoogle Scholar
  22. Lin RZ, Dreyzin A, Aamodt K, Dudley AC, Melero-Martin JM (2011) Functional endothelial progenitor cells from cryopreserved umbilical cord blood. Cell Transpl 20:515–522. CrossRefGoogle Scholar
  23. Lu XM, Proctor SJ, Dickinson AM (2008) The effect of cryopreservation on umbilical cord blood endothelial progenitor cell differentiation. Cell Transpl 17:1423–1428. CrossRefGoogle Scholar
  24. Mieno S, Clements RT, Boodhwani M, Sodha NR, Ramlawi B, Bianchi C, Sellke FW (2008) Characteristics and function of cryopreserved bone marrow-derived endothelial progenitor cells. Ann Thorac Surg 85:1361–1366. CrossRefGoogle Scholar
  25. Mukai N et al (2008) A comparison of the tube forming potentials of early and late endothelial progenitor cells. Exp Cell Res 314:430–440. CrossRefGoogle Scholar
  26. Ohtani K et al (2004) Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells. Circulation 110:2444–2452. CrossRefGoogle Scholar
  27. Papa ND et al (2006) Bone marrow endothelial progenitors are defective in systemic sclerosis. Arthritis Rheum 54:2605–2615. CrossRefGoogle Scholar
  28. Papasavvas E et al (2012) Increased CD34(+)/KDR(+) cells are not associated with carotid artery intima-media thickness progression in chronic HIV-positive subjects. Antivir Ther 17:557–563. CrossRefGoogle Scholar
  29. Peichev M et al (2000) Expression of VEGFR-2 and AC133 by circulating human CD34 + cells identifies a population of functional endothelial precursors. Blood 95:952–958Google Scholar
  30. Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM (2009) Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell 4:62–72. CrossRefGoogle Scholar
  31. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, Duan J, Imaizumi T (2001) Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 103:897–903. CrossRefGoogle Scholar
  32. Sukmawati D, Tanaka R (2015) Introduction to next generation of endothelial progenitor cell therapy: a promise in vascular medicine. Am J Transl Res 7:411–421Google Scholar
  33. Tamarat R et al (2004) Impairment in ischemia-induced neovascularization in diabetes: bone marrow mononuclear cell dysfunction and therapeutic potential of placenta growth factor treatment. Am J Pathol 164:457–466CrossRefGoogle Scholar
  34. Tepper OM et al (2002) Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation 106:2781–2786. CrossRefGoogle Scholar
  35. Teraa M et al (2013) Bone marrow alterations and lower endothelial progenitor cell numbers in critical limb ischemia patients. PLOS ONE 8:e55592. CrossRefGoogle Scholar
  36. Tsai S, Butler J, Rafii S, Liu B, Kent KC (2009) The role of progenitor cells in the development of intimal hyperplasia. J Vasc Surg 49:502–510. CrossRefGoogle Scholar
  37. Umemura T et al (2008) Aging and hypertension are independent risk factors for reduced number of circulating endothelial progenitor cells. Am J Hypertens 21:1203–1209. CrossRefGoogle Scholar
  38. Urbich C, Dimmeler S (2004) Endothelial progenitor cells characterization and role in vascular biology. Circ Res 95:343–353. CrossRefGoogle Scholar
  39. Vanneaux V et al (2010) In vitro and in vivo analysis of endothelial progenitor cells from cryopreserved umbilical cord blood: are we ready for clinical application? Cell Transpl 19:1143–1155. CrossRefGoogle Scholar
  40. Vasa M et al (2001) Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:e1–e7. CrossRefGoogle Scholar
  41. von Bomhard A, Elsässer A, Ritschl LM, Schwarz S, Rotter N (2016) Cryopreservation of endothelial cells in various cryoprotective agents and media-vitrification versus slow freezing methods. PLoS ONE 11:e0149660. CrossRefGoogle Scholar
  42. Wall ME, Bernacki SH, Loboa EG (2007) Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells. Tissue Eng 13:1291–1298. CrossRefGoogle Scholar
  43. Wu J et al (2012) Optimization of cryopreservation procedures for porcine endothelial progenitor cells. J Biosci Bioeng 113:117–123. CrossRefGoogle Scholar
  44. Yang C, Zhang ZH, Li ZJ, Yang RC, Qian GQ, Han ZC (2004) Enhancement of neovascularization with cord blood CD133 + cell-derived endothelial progenitor cell transplantation. Thromb Haemost 91:1202–1212. CrossRefGoogle Scholar
  45. 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. CrossRefGoogle Scholar
  46. Yin Y, Liu H, Wang F, Li L, Deng M, Huang L, Zhao X (2015) Transplantation of cryopreserved human umbilical cord blood-derived endothelial progenitor cells induces recovery of carotid artery injury in nude rats. Stem Cell Res Ther 6:37. CrossRefGoogle Scholar
  47. Young PP, Hofling AA, Sands MS (2002) VEGF increases engraftment of bone marrow-derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients. Proc Natl Acad Sci USA 99:11951–11956. CrossRefGoogle Scholar
  48. Yu D, Chen W, Ren J, Zhang T, Yang K, Wu G, Liu H (2014) VEGF-PKD1-HDAC7 signaling promotes endothelial progenitor cell migration and tube formation. Microvasc Res 91:66–72. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang UniversityBeijingPeople’s Republic of China
  2. 2.Beijing Advanced Innovation Centre for Biomedical EngineeringBeihang UniversityBeijingPeople’s Republic of China
  3. 3.National Research Center for Rehabilitation Technical AidsBeijingPeople’s Republic of China

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