Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters, Producing High Levels of VEGF and Forming Capillaries

Francesco De Francesco,Virginia Tirino,Vincenzo Desiderio,Giuseppe Pirozzi,Giuseppe Ferraro,Guido Libondi,Francesco D'Andrea,Alfredo De Rosa,Mariateresa Giuliano,Gianpaolo Papaccio,Bernadette Breant

doi:10.1371/journal.pone.0006537

Abstract

BackgroundHuman adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.Methodology/Principal FindingsIn this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34+/CD90+ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34+/CD90+ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31+/VEGF+/Flk-1+) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.Conclusions/SignificanceOur results demonstrate, for the first time, that CD34+/CD90+ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

Highlights

Stem cells are undifferentiated cells that undergo multiple, sequential cell divisions, that have the ability to renew themselves, and that, in the adult, are responsible for repair and repopulating damaged tissues [1]
Our study has shown the following results and novelties with respect to the existing literature on this topic: (i) two different populations can be detected within the stromal vascular fraction (SVF) of human adipose tissue: mesenchymal stem cells expressing CD90, CD29, CD44, CD105, CD117, and CD34; and endothelial progenitor cells, expressing CD34, CD90, CD44, and CD54; (ii) CD34+/CD90+ cells are capable of differentiating into endothelial cells (CD31+/ VEGF+/Flk-1+) and, when stimulated with an adipogenic medium, into adipocytes; (iii) these cells are capable of forming sphere clusters, when grown in free-floating conditions, and capillary-like structures, when challenged on methylcellulose, as substantiated by production of VEGF
Flow cytometry assay At day 0, antigenic analysis of cells belonging to both mammary and abdominal regions showed that different cell types were detectable within the SVF; these were positive for CD29 (b1integrin) (89%), CD34 (33%), CD90 (Thy-1) (52%), CD117 (10%), CD105 (12%), and CD133 (4%); and for differentiated cell-associated markers such as CD31 (9%), CD44 (5%) CD54 (9%), and Flk-1 (VEGFR-2) (32%)

Summary

Introduction

Stem cells are undifferentiated cells that undergo multiple, sequential cell divisions, that have the ability to renew themselves, and that, in the adult, are responsible for repair and repopulating damaged tissues [1]. They are the natural and direct source from which stable differentiated cells can form an adult tissue. Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). It is an accessible site producing a considerable amount of stem cells

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Conclusion