Fibroblast Growth Factors Regulate Stem Cell Functioning In Vitro and in Vivo.

Fibroblast Growth Factors (FGF) are a large family of signaling molecules widely involved in tissue development, maintenance and repair. Little is known about the role of FGF/FGF-receptor signaling in the regulation of adult hematopoietic stem cells (HSC). In this study, we assessed the potential of exogenously added FGF-1/2, or retrovirally overexpressed FGF-1 to preserve HSC function in vitro and in vivo.

First, we demonstrate that in vitro culture of unfractionated mouse bone marrow cells, in serum-free medium, supplemented with FGF-1 or FGF-2 or FGF-1 + 2 resulted in the robust generation of long-term repopulating (LTR) HSCs. Cultures were maintained for 12 weeks and during that time in vivo competitive reconstitution assays were performed. Stem cell activity was detectable at 3, 5, and 8 weeks after initiation of culture, but lost after 12 weeks. However, whereas 3 and 5 week cultured cells provided radioprotection in non-competitive assays, animals transplanted with 8 or 12 week cultured cells succumbed due to bone marrow failure. So far, we have been unable to expand single, highly purified Lin⁻Sca-1⁺c-Kit⁺ using FGF-1 + 2. Consequently, we speculated that essential intermediate cell populations or signals are required for FGF-induced stem cell conservation. To test this we cultured highly purified CD45.1 Lin⁻Sca-1⁺c-Kit⁺ cells in a co-culture with CD45.2 unfractionated BM. Co-cultured cells were transplanted after 5 weeks in lethally irradiated recipients, and CD45.1 chimerism levels were assessed. High levels of CD45.1 chimerism confirmed that Lin⁻Sca-1⁺c-Kit⁺ cells require an accessory signal in addition to FGF to induced stem cell activity in vitro. We subsequently tested stem cell potential of cells cultured in FGF-1 + 2 for 5 weeks, with the addition of SCF + IL-11 + Flt3L for the last 2, 4 or 7 days. Cell numbers increased with increasing time of growth factor presence. However, only when growth factors were present for 2 days engraftment of cultured cells in a competitive repopulation assay was increased 3.5-fold.

Finally, we show by immunohistochemistry that ~10% of freshly isolated Lin⁻Sca-1⁺c-Kit⁺ expresses high levels of FGF-1. Retroviral overexpression of FGF-1 in stem cells resulted in increased growth potential and sustained clonogenic activity in vitro. Upon transplantation of transduced stem cells, FGF-1 overexpression resulted in increased white blood cell counts 4 weeks post-transplant compared to control animals. Most notable was a marked granulocytosis in FGF-1 overexpressing recipients Our results reveal FGF as an important regulator of HSC signaling and homeostasis. Importantly, in the presence of FGF stem cells can be maintained in vitro for 2 months. These findings open novel avenues for in vitro manipulation of stem cells for future clinical therapies.