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TISSUE-SPECIFIC STEM CELLS

Fibroblast Growth Factor-1 and -2 Preserve Long-Term Repopulating Ability of Hematopoietic Stem Cells in Serum-Free Cultures

^a Department of Cell Biology, Section Stem Cell Biology, University Medical Centre Groningen, Groningen, The Netherlands;
^b Department of Hematology, University Medical Centre Groningen, Groningen, The Netherlands

Key Words. Hematopoietic stem cells • Serum-free culture • Preservation • Fibroblast growth factors

Correspondence: Prof. Gerald de Haan, Ph.D., Department of Cell Biology, Section Stem Cell Biology, University Medical Centre Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands. Telephone: + 31 503632722; Fax: + 31 503637477; e-mail: g.de.haan{at}med.umcg.nl

Received September 8, 2005; accepted for publication March 5, 2006.

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin^–Sca-1⁺c-Kit⁺(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.

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