Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties


Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

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This work was supported by grants from the PROMEX STIFTUNG FUR DIE FORSCHUNG foundation that we deeply thank. We thank all people involved in Animal Platform, CRP2 - UMS 3612 CNRS - US25 Inserm-IRD – Faculté de Pharmacie de Paris, Université Paris Descartes, Paris, France.

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ER performed the experiments, acquired and analysed the data; and prepared manuscript. SCP, AS, AL, PB, BD, NCB, NG, AB performed the experiments and acquired the data. RC analysed data and performed statistical analysis. IB, CLG and PG participated to coordination and edited the manuscript. DMS analysed the data, conceived the study, participated in its coordination and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to David M. Smadja.

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Rossi, E., Poirault-Chassac, S., Bieche, I. et al. Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties. Stem Cell Rev and Rep 15, 590–600 (2019).

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  • Endothelial progenitor
  • Endothelial colony forming cells
  • ECFC
  • CD133
  • Prominin-1
  • Stem cells
  • Stemness
  • Angiogenesis