Annals of Biomedical Engineering

, Volume 43, Issue 10, pp 2552–2568 | Cite as

Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro

  • Erica B. Peters
  • Betty Liu
  • Nicolas Christoforou
  • Jennifer L. West
  • George A. Truskey
Article

Abstract

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and similar effects as HVPs upon total EPC tubule length (p = 0.95) and number of branch points (p = 0.93). In a reductionist coculture system, MNCs served as pro-angiogenic mural cells by supporting EPC network formation to a significantly greater extent than HVP cocultures, by day 14 of coculture, as evidenced through EPC total tubule length (p < 0.0001) and number of branch points (p < 0.0001). Our findings are significant as we demonstrate mural cell progenitors can be isolated from umbilical cord blood and develop culture conditions to support their use in microvascular tissue engineering applications.

Keywords

Angiogenesis Vasculogenesis Microvessel formation Umbilical cord blood Progenitor cells Tissue engineering 

Abbreviations

EC

Endothelial cell

EndMT

Endothelial-to-mesenchymal transition method for isolating MPCs from cord blood

EPC

Endothelial progenitor cell

HUVEC

Human umbilical vein-derived endothelial cell

HVP

Human vascular pericyte

MNC

Mononuclear cell

MPC

Mesenchymal progenitor cell

MSC

Mesenchymal stem cell

SMC

Smooth muscle cell

SS-MNC

Spindle-shaped mononuclear cell

TM

Traditional method for isolating MPCs from cord blood

Notes

Acknowledgements

This work was supported in part by NIH Grant HL88825. E.B.P. was supported by a National Science Foundation Graduate Research Fellowship. This work was also supported by the Flight Attendant Medical Research Institute (Young Clinical Scientist Award to N.C.).

Supplementary material

10439_2015_1301_MOESM1_ESM.docx (14.9 mb)
Supplementary material 1 (DOCX 15212 kb)

References

  1. 1.
    Adams, R. H., and K. Alitalo. Molecular regulation of angiogenesis and lymphangoigenesis. Nat. Rev. 8:464–478, 2007.CrossRefGoogle Scholar
  2. 2.
    Armulik, A., A. Abramsson, and C. Betsholtz. Endothelial/pericyte interactions. Circ. Res. 97:512–523, 2005.CrossRefPubMedGoogle Scholar
  3. 3.
    Armulik, A., G. Genové, and C. Betsholtz. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell. 21:193–215, 2011.CrossRefPubMedGoogle Scholar
  4. 4.
    Attar, A., A. Ghalyanchi Langeroudi, A. Vassaghi, I. Ahrari, M. K. Maharlooei, and A. Monabati. Role of CD271 enrichment in the isolation of mesenchymal stromal cells from umbilical cord blood. Cell Biol. Int. 37:1010–1015, 2013.CrossRefPubMedGoogle Scholar
  5. 5.
    Bhang, S. H., S. Lee, J. Y. Shin, T. J. Lee, and B. S. Kim. Transplantation of cord blood mesenchymal stem cells as spheroids enhances vascularization. Tissue Eng. Part A. 18:2138–2147, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Birbrair, A., T. Zhang, Z. M. Wang, M. L. Messi, A. Mintz, and O. Delbono. Type-1 pericytes participate in fibrous tissue deposition in aged skeletal muscle. Am J Physiol Cell Physiol. 305:C1098–C1113, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Blocki, A., Y. Wang, M. Koch, P. Peh, S. Beyer, P. Law, J. Hui, and M. Raghunath. Not all MSCs can act as pericytes: functional in vitro assays to distinguish pericytes from other mesenchymal stem cells in angiogenesis. Stem Cells Dev. 22:2347–2355, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bourghardt Peebo, B., P. Fagerholm, C. Traneus-Röckert, and N. Lagali. Time-lapse in vivo imaging of corneal angiogenesis: the role of inflammatory cells in capillary sprouting. Invest Ophthalmol. Vis. Sci. 10:3060–3068, 2011.CrossRefGoogle Scholar
  9. 9.
    Butler, J. M., H. Kobayashi, and S. Rafii. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiogenic factors. Nat. Rev. Cancer 10:138–146, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Caplan, A. I. All MSCs are pericytes? Cell Stem Cell 3:229–230, 2008.CrossRefPubMedGoogle Scholar
  11. 11.
    Chen, W. C., T. S. Park, I. R. Murray, L. Zimmerlin, L. Lazzari, J. Huard, and B. Péault. Cellular kinetics of perivascular MSC precursors. Stem Cells Int. 2013:983059, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Covas, D. T., R. A. Panepucci, A. M. Fontes, W. A. Jr., M. D. Orellana Silva, M. C. Freitas, L. Neder, A. R. Santos, L. C. Peres, M. C. Jamur, and M. A. Zago. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts. Exp. Hematol. 36:642–654, 2008.CrossRefPubMedGoogle Scholar
  13. 13.
    Crisostomo, P. R., M. Wang, G. M. Wairiuko, E. D. Morrell, A. M. Terrell, P. Seshadri, U. H. Nam, and D. R. Meldrum. High passage number of stem cells adversely affects stem cell activation and myocardial protection. Shock 26:575–580, 2006.CrossRefPubMedGoogle Scholar
  14. 14.
    Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. J. Prockop, and E. Horwitz. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8:315–317, 2006.CrossRefPubMedGoogle Scholar
  15. 15.
    Evensen, L., D. R. Micklem, A. Blois, S. V. Berge, N. Aarsaether, A. Littlewood-Evans, J. Wood, and J. B. Lorens. Mural cell associated VEGF is required for organotypic vessel formation. PLoS One 4:e5798, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ferrara, N., and R. S. Kerbel. Angiogenesis as a therapeutic target. Nature 438:967–974, 2005.CrossRefPubMedGoogle Scholar
  17. 17.
    Foo, S. S., C. J. Turner, S. Adams, A. Compagni, D. Aubyn, N. Kogata, P. Lindblom, M. Shani, D. Zicha, and R. H. Adams. Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly. Cell 124:161–173, 2006.CrossRefPubMedGoogle Scholar
  18. 18.
    Herbert, S. P., and D. Y. Stainier. Molecular control of endothelial cell behavior during blood vessel morphogenesis. Nat. Rev. Mol. Cell Biol. 12:551–564, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hirschi, K. K., D. A. Ingram, and M. C. Yoder. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 28:1584–1595, 2008.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hirvonen, T., H. Suila, S. Tiitinen, S. Natunen, M. L. Laukkanen, A. Kotovuori, M. Reinman, T. Satomaa, K. Alfthan, S. Laitinen, K. Takkinen, J. Räbinä, and L. Valmu. Production of a recombinant antibody specific for I blood group antigen, a mesenchymal stem cell marker. Bioresour. Open Access. 2:336–345, 2013.CrossRefGoogle Scholar
  21. 21.
    Ingram, D. A., L. E. Mead, H. Tanaka, V. Meade, A. Fenoglio, K. Mortell, K. Pollok, M. J. Ferkowicz, D. Gilley, and M. C. Yoder. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104:2752–2760, 2004.CrossRefPubMedGoogle Scholar
  22. 22.
    Kleinman, H. K., M. L. McGarvey, L. A. Liotta, P. G. Robey, K. Tryggvason, and G. R. Martin. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193, 1982.CrossRefPubMedGoogle Scholar
  23. 23.
    Korff, T., S. Kimmina, G. Martiny-Baron, and H. G. Augustin. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 15:447–457, 2001.CrossRefPubMedGoogle Scholar
  24. 24.
    Kretlow, J. D., Y. Q. Jin, W. Liu, W. J. Zhang, T. H. Hong, G. Zhou, L. S. Baggett, A. G. Mikos, and Y. Cao. Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol. 9:60, 2008.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Laitinen, A., J. Nystedt, and S. Laitinen. The isolation and culture of human cord blood-derived mesenchymal stem cells under low oxygen conditions. Methods Mol. Biol. 698:63–73, 2011.CrossRefPubMedGoogle Scholar
  26. 26.
    Lee, O. K., T. K. Kuo, W. M. Chen, K. D. Lee, S. L. Hsieh, and T. H. Chen. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675, 2004.CrossRefGoogle Scholar
  27. 27.
    Liu, Y., S. H. Teoh, M. S. Chong, C. H. Yeow, R. D. Kamm, M. Choolani, and J. K. Chan. Contrasting effects of vasculogenic induction upon biaxial bioreactor stimulation of mesenchymal stem cells and endothelial progenitor cells cocultures in three-dimensional scaffolds under in vitro and in vivo paradigms for vascularized bone tissue engineering. Tissue Eng. Part A. 19:893–904, 2013.CrossRefPubMedGoogle Scholar
  28. 28.
    Markov, V., K. Kusumi, M. G. Tadesse, D. A. William, D. M. Hall, V. Lounev, A. Carlton, J. Leonard, R. I. Cohen, E. F. Rappaport, and B. Saitta. Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev. 16:53–73, 2007.CrossRefPubMedGoogle Scholar
  29. 29.
    Medici, D., and R. Kalluri. Endothelial-mesenchymal transition and its contribution to the emergence of stem cell phenotype. Semin. Cancer Biol. 22:379–384, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Medici, D., E. M. Shore, V. Y. Lounev, F. S. Kaplan, R. Kalluri, and B. R. Olsen. Conversion of vascular endothelial cells into multipotent stem-like cells. Nat. Med. 16:1400–1406, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Melero-Martin, J. M., M. E. De Obaldia, S. Y. Kang, Z. A. Khan, L. Yuan, P. Oettgen, and J. Bischoff. Engineering robust and functional vascular networks in vivo with human adult and cord blood derived progenitor cells. Circ. Res. 103:194–202, 2008.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Melero-Martin, J. M., Z. A. Khan, A. Picard, X. Wu, S. Paruchuri, and J. Bischoff. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood 109:4761–4768, 2007.CrossRefPubMedGoogle Scholar
  33. 33.
    Moonen, J. R., G. Krenning, M. G. Brinker, J. A. Koerts, M. J. van Luyn, and M. C. Harmsen. Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny. Cardiovasc. Res. 86:506–515, 2010.CrossRefPubMedGoogle Scholar
  34. 34.
    Novosel, E. C., C. Kleinhans, and P. J. Kluger. Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 63:300–311, 2011.CrossRefPubMedGoogle Scholar
  35. 35.
    Orr, A. W., C. A. Elzie, D. F. Kucik, and J. E. Murphy-Ullrich. Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration. J. Cell Sci. 116:2917–2927, 2003.CrossRefPubMedGoogle Scholar
  36. 36.
    Pedersen, T. O., A. L. Blois, Y. Xue, Z. Xing, M. Cottler-Fox, I. Fristad, K. N. Leknes, J. B. Lorens, and K. Mustafa. Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells. J. Tissue Eng. 3:2041731412443236, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Peters, E. B., N. Christoforou, K. W. Leong, and G. A. Truskey. Comparison of mixed and lamellar coculture spatial arrangements for tissue engineering capillary networks in vitro. Tissue Eng. Part A. 19:697–706, 2013.CrossRefPubMedGoogle Scholar
  38. 38.
    Roura, S., J. R. Bagó, C. Soler-Botija, J. M. Pujal, C. Gálvez-Montón, C. Prat-Vidal, A. Llucià-Valldeperas, J. Blanco, and A. Bayes-Genis. Human umbilical cord blood-derived mesenchymal stem cells promote vascular growth in vivo. PLoS One 7:e49447, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Saik, J. E., D. J. Gould, A. H. Keswani, M. E. Dickinson, and J. L. West. Biomimetic hydrogels with immobilized ephrinA1 for therapeutic angiogenesis. Biomacromolecules 12:2715–2722, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Senger, D. R., and G. E. Davis. Angiogenesis. Cold Spring Harb Perspect Biol 3:a005090, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Sharma, R. R., K. Pollock, and A. Hubel. Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion 54:1418–1437, 2014.CrossRefPubMedGoogle Scholar
  42. 42.
    Stanevsky, A., G. Goldstein, and A. Nagler. Umbilical cord blood transplantation: pros, cons and beyond. Blood Rev 23:199–204, 2009.CrossRefPubMedGoogle Scholar
  43. 43.
    ten Dijke, P., and H. M. Arthur. Extracellular control of TGFbeta signaling in vascular development and disease. Nat. Rev. Mol. Cell. Biol. 8:857–869, 2007.CrossRefPubMedGoogle Scholar
  44. 44.
    Wagner, W., S. Bork, P. Horn, D. Krunic, T. Walenda, A. Diehlmann, V. Benes, J. Blake, F. X. Huber, V. Eckstein, P. Boukamp, and A. D. Ho. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One 4:e5846, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Yee, D., D. Hanjaya-Putra, V. Bose, E. Luong, and S. Gerecht. Hyaluronic acid hydrogels support cord-like structures from endothelial colony-forming cells. Tissue Eng. Part A. 17:1351–1361, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Zarem, H. A. The microcirculatory events within full-thickness skin allografts (homo-grafts) in mice. Surgery 66:392–397, 1969.PubMedGoogle Scholar
  47. 47.
    Zhang, X., M. Hirai, S. Cantero, R. Ciubotariu, L. Dobrila, A. Hirsh, K. Igura, H. Satoh, I. Yokomi, T. Nishimura, S. Yamaguchi, K. Yoshimura, P. Rubinstein, and T. A. Takahashi. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J. Cell Biochem. 112:1206–1218, 2011.CrossRefPubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2015

Authors and Affiliations

  • Erica B. Peters
    • 1
  • Betty Liu
    • 1
  • Nicolas Christoforou
    • 1
  • Jennifer L. West
    • 1
  • George A. Truskey
    • 1
  1. 1.Duke UniversityDurhamUSA

Personalised recommendations