Properties of MSC Derived Stromal Cell Lines Developed from Bone Marrow of TNF Knockout Mice.

Mesenchymal stem cells (MSC) forming adherent stromal cell lines have been revealed in suspension fraction (SF) of 70+ weeks old long-term bone marrow culture (LTBMC) of TNF knockout mice.

The proportion of CD45+ cells in such old cultures decreased significantly while cellularity of SF increased, accompanied by the first successful generation of cell lines. More than 100 cell lines were established.

Almost all cell lines were Sca1 positive, expressed SDF1, had fibroblast-like morphology and expressed collagens type II and IV. Most of cell lines could maintain hematopoiesis (CFU-S, CFU-GM and CAFC production) for 10 weeks. Some of the cell lines were able to maintain growth without differentiation of murine ES cells and some expressed LIF or LIF-receptor, VEGF and FGF1. All cell lines produced hematopoietic growth factors - their supernatants stimulated growth of CFU-GM in semisolid media. The expression of SCF was revealed in all cell lines tested for growth factors production.

Quantitative RT-PCR analysis revealed that the expression of hematopoietic growth factors - M-CSF, G-CSF and TPO increased sometimes up to 6 times in comparison with the expression of these factors in adherent cell layer (ACL) of LTBMC. The expression of N-cadherin - one of the main regulators of hematopoietic stem cells (HSC) in niche - increased 7–32 fold in 7 tested cell lines regarding the expression of this RNA in ACL of LTBMC. Whereas the expression level of N-cadherin was lower in wild type bone marrow, NIH3T3 fibroblasts and MS5 cells, maintaining the hematopoiesis and CAFC growth, showed the expression on the higher level than in ACL. The expression of ICAM 1, the molecule also regulating HSC, was reduced in all cell lines. The expression level of genes participating in activating of hematopoietic stem cells in niche such as Jagged, Dll 1 and Ang1 more than halved in 5 out of 7 tested lines. The production of Wnt 5a RNA fluctuated from 0,5 to 12 fold and did not correlate with any other changes in gene expression and the ability of cell lines to maintain hematopoiesis. The level of Ihh, Shh was downregulated in cell lines.

The cell line 26dD3 developed in the media containing dexamethasone demonstrated 70 fold increased expression of Kirre - gene supporting HSC but there was no difference in maintaining hematopoiesis and ES cells. High expression of human homolog of this gene (NEPH2) was known to be detected in brain only. At the same time this cell line expressed tubulin b3. These data support a model of lineage specification in which unilineage commitment is prefaced by a “promiscuous” phase of multilineage locus activation. Thus the developed stromal cell lines are not terminally differentiated and use different pathways to maintain hematopoiesis. The TNF deficiency leads to dramatic alterations in regulation of MSC. Mechanisms of this regulation should be scrutinized.