Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1-155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.

Background/Aims: The influence of cytostatic medicines on mesenchymal stem cells (MSC) and their progeny, fibroblastic colony-forming units (CFU-F), was investigated. Methods: Mice were treated with busulfan, cyclophosphamide, cytarabine, methotrexate and bortezomib, as used in clinical practice. MSC and CFU-F were analyzed 3 days and 6 weeks after the treatment termination. To estimate MSC numbers, the ectopic foci formation method was used. Briefly, a donor bone marrow plug was transplanted under the renal capsule of a syngeneic animal, leading to ectopic foci formation. The systemic response of the hematopoietic microenvironment to these drugs was studied using the same method applied to recipients pretreated with the medicines. Results: CFU-F concentration was halved in the bone marrow of mice treated with busulfan, methotrexate and cyclophosphamide, and was not restored for the next 6 weeks. Proliferative potential and differentiation abilities of MSC were not affected by these medicines. The enlargement of foci size in mice treated with cytostatic agents was not conditioned by MSC, but by more mature stromal precursor cells. Conclusions: Cytostatic medicines affect stromal precursors in 2 ways: they decrease CFU-F concentration in the ‘steady-state’ bone marrow, while stimulating growth of the stromal microenvironment during its de novo formation. MSC are not sensitive to the cytostatic agents used.

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The hierarchy of stromal precursor cells was studied. Changes in the number of fibroblast CFU in foci forming in irradiated recipients were analyzed. These precursor cells are most close known descendants of mesenchymal stem cells, while inducible precursors rank lower in the hierarchy and are the cells directly enlarging the hemopoietic area in irradiated recipients.

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo.