HB-EGF/HER -1 SIGNALLING IN BONE MARROW MESENCHYMAL STEM CELLS: INDUCING CELL EXPANSION AND PREVENTING REVERSIBLY MULTI-LINEAGE DIFFERENTIATION

Epidermal growth factor receptor-1 (EGFR-1/HER- 1/ErbB) regulates proliferation and cell fate during epidermal development. HER-1 is activated by several EGF-family ligands including heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogenic and chemotactic molecule that participates to tissue repair, tumour growth and other tissue-modelling phenomena, such as angiogenesis and fibrogenesis. In many of the processes in which HB-EGF is often involved, mesenchymal stem cells (MSCs), the precursors of different mes- enchymal tissues, have a role. We have studied whether the HB-EGF/HER -1 system is expressed in human MSCs and which role it plays in MSC biology. MSCs have been generated from bone marrow aspirates of healthy donors, recruited after informed consent, and expanded in com- plete DMEM medium (15% FCS). MSCs have been characterized by immunophenotype and in vitro multilineage differentiation. The expression of HB-EGF, HER-1 and HER-4 (the other receptor for HB-EGF) has been studied by flow-cytometry and RT-PCR. Then we have studied the short- and long-term effect of HB-EGF on MSC proliferation and multilineage differentiation by specific assays and differentiation-specific gene expression by quantitative RT-PCR. We have found that MSCs normally express HER-1, but not HB-EGF or HER-4. Under the effect of HB- EGF, MSCs proliferate more rapidly and persistently, without undergoing spontaneous differentiation. This effect occurs in a dose-dependent fashion, and is specific, direct, long-lasting, comparable to other growth factors such as bFGF, and HER-1-mediated, as it is inhibited by anti-HER- 1 and anti-HB-EGF blocking antibodies. The effect is tighly controlled because surface HER-1 is down-regulated after interaction with HB-EGF: this occurs rapidly but reversibly, because HER-1 RNA is still synthetized and leads to the re-expression of surface HER-1 a few hours after HB-EGF removal from culture. By contrast, HER-1 expression is permanently lost during MSC differentiation into mesenchymal cell lineages. Moreover, HB-EGF reversibly prevents adipogenic, osteogenic and chondro- genic differentiation induced with specific media, by pre- serving MSC potential. This study provides the first evidence that HB-EGF/HER-1 signalling is mitogenic for MSCs and may prevent reversibly their differentiation, leading to self-renewing rather than differentiative cell divisions. The rapid ex-vivo MSC expansion and down-regulation of their sensitivity to physiological differentiation agents could represent a valid alternative to other factors, such as bFGF, with an advantage in terms of MSC differentiation potential, in situ recruitment and proliferation, and therefore of in vivo transplant efficiency. It has to be investigated whether the HB-EGF/HER-1 signalling may contribute in vivo to maintain a broad, proliferating pool of undifferentiated MSC, thus ensuring the regenerative process or the efficient angiogenesis to neoplastic growth. The use of HB-EGF inhibitors could have a role in these conditions.