cDNA cloning of FRIL, a lectin from Dolichos lablab, that preserves hematopoietic progenitors in suspension culture

Abstract

Ex vivo culture of hematopoietic stem cells is limited by the inability of cytokines to maintain primitive cells without inducing proliferation, differentiation, and subsequent loss of repopulating capacity. We identified recently in extracts of kidney bean and hyacinth bean a mannose-binding lectin, called FRIL, and provide here evidence that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transfected with Flt3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose-binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone is able to preserve cells for 1 month without medium changes. In vitro progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast-like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL's clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation, and ex vivo cultures used for expansion and stem cell gene therapy currently are being explored.

Figure 1

Fractionation of purified hyacinth bean FRIL by SDS/PAGE and amino-terminal amino acid sequences of the constituent polypeptides.

Figure 2

Amino acid sequence alignments of the determined amino acid sequence of the hyacinth bean lectin [see Gowda et al. (7)], the derived amino acid sequence obtained from the FRIL cDNA, and the sequences of two peptides determined with the purified protein obtained from the same beans. The underlined tripeptides represent potential N-linked glycosylation sites.

Figure 3

Colonies derived from cord blood cells cultured in FRIL. Blast-like colonies formed in methylcellulose colony assays after exposure of cord blood mononuclear cells to FRIL for 3 weeks (A) or 4 weeks (B). Colonies that formed after 3 weeks of FRIL generated yet more blast-like colonies (C).

Figure 4

Serial replating of progenitors cultured in FRIL. (A) CB mnc were cultured for 3 weeks in suspension culture in FRIL (solid box). (B) Harvested cells were assessed for progenitor capacity in methylcellulose colony assay (striped box) for 6 weeks. (C) Cells harvested from the colony assay were replated in a second colony assay for an additional 4 weeks. Progenitor frequencies were determined both for cells after 3 weeks of suspension culture and for cells harvested after an additional 6 weeks of methylcellulose culture (indicated by arrows). ND, not detected.