Abstract
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.
Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland