IL-7 and immobilized Kit-ligand stimulate serum- and stromal cell-free cultures of precursor B-cell lines and clones

Yohei Kawano, Georg Petkau, Ingrid Wolf, Julia Tornack, Fritz Melchers, Apollo-University Of Cambridge Repository, Apollo-University Of Cambridge Repository
2019
Long-term proliferating, D H J H -rearranged mouse precursor B-cell lines have previously been established in serum-and IL-7-containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre-B cells to undefined factors, hampering accurate analyses of ligand-dependent signaling, which controls pre-B cell proliferation, survival, residence and migration. Here, we describe a novel serum-free, stromal cell-free culture system, which allows us to establish and
more » ... to establish and maintain pre-B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit-ligand, also called stem cell factor, produced as a kit-ligand-Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre-B cells in an optimal density in plate-bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre-B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow. Keywords: IL-7 r Kit-ligand r Pre-B cell r Proliferation r Serum-free r Stem cell factor r Stromal cell-free Additional supporting information may be found in the online version of this article at the publisher's web-site occupy distinct niches. Niches for HSC are found in perivascular, sinusoidal areas near endothelial cells within bone marrow, while early B-cell progenitors and precursors have been localized in sub-endosteal regions, near the inner bone surface, in the neighborhood of osteoblasts [4, 5] . HSC and their progeny up to pre-BI cells express membrane-bound tyrosine kinase c-kit, which is involved in the regulation of cell proliferation [6] . Kit-ligand (kit-L), also called stem cell factor, is provided in the HSC niche by endothelial cells, in CLP/pro-pre-B-and pre-BI cell niches by mesenchymally-derived osteoblasts, i.e. non-hematopoietic stromal cells. Such stromal cells have been established as cell lines
doi:10.17863/cam.41254 fatcat:qf3k4gqm75cz3cwtifzlhjwqv4