Specific dose-response effects of TGF-beta1 on developmentally distinct hematopoietic stem/progenitor cells from human umbilical cord blood.

INTRODUCTION: Transforming Growth Factor-beta1 is known to maintain primitive human hematopoietic stem/progenitor cells in a quiescent state. However, its specific role in the control of distinct progenitor cell types needs to be further elucidated. In this study, we have investigated the dose-response effect of TGF-beta1 on progenitors ranging from primitive high proliferative potential (HPP)-Mix, -GM or -BFU-E to later BFU-E, CFU-G or CFU-M. MATERIALS AND METHODS: A clonal semi-solid assay has been used to analyze the effects of a TGF-beta1 blocking antibody (anti-TGF-beta1) or that of active TGF-beta1 added to the medium at concentrations from 10-3000 pg/ml, on these different hematopoietic stem/progenitor cell types. RESULTS AND CONCLUSION: A preferential growth inhibitory effect on the earlier progenitors was observed when low concentrations of TGF-beta1 (10-300 pg/ml) were used. Concentrations of 10-30 pg/ml TGF-beta1 were sufficient to inhibit 90% of the primitive multipotent HPP-Mix, while 100-300 pg/ml TGF-beta1 were required to inhibit 70% of the bipotent HPP-GM and early HPP-BFU-E. TGF-beta1 did not significantly inhibit or activate the growth of later CFU-G and CFU-M, even when added at concentrations 10-100 fold higher. In contrast, a significant growth-inducing effect of very low TGF-beta1 concentrations (< or =30 pg/ml) on a subset of later BFU-E was observed and cannot be explained by a switch of early into later BFU-E. These results emphasize the polyfunctional role of TGF-beta1 in the regulation of hematopoiesis and the need for low, physiological concentrations of TGF-beta1, when studying both the stem cell compartment and more mature progenitor cell subpopulations.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.