The Proliferative Ability of Adult Bone Marrow Native Mesenchymal Progenitors (NMP) Is Early Altered during In Vitro Passaging.

The rarity of adult bone marrow native mesenchymal stem/progenitor cells and the difficulty in characterizing them in fresh organs renders their amplification necessary before use. Little is known about NPM and the correlation between the properties of amplified cells and those of NPM remains unknown.

To study the NPM, the sorted fresh CD45⁻ CD14⁻ CD73⁺ cell subsets (Boiret et al., Exp. Hematol. 2005) were seeded in a cloning assay. We studied the proliferation ability of mesenchymal cells (MC) from bone marrow (BM) grafts (n=35), filtered hematons (H) (n=10), and spongious bone from femoral head (FH) (n=131), until P2-3 (mean culture duration: 6 weeks in IMDM 10% FCS 1% L-Glutamin). The median age of patients was 38±3 years ranging from 21 to 75 years.

Seeding of fresh FH CD45⁻ CD14⁻ CD73⁺ at very low densities showed that about 0.6% of these cells adhered with a fibroblast-like morphology. More than 60 % of them were NMP and capable of cloning, with a very heterogeneous proliferation amplitude. In the first 10 days, cell proliferation represented a mean of 3.8 doubling population (DP). Addition of EGF and PDGF accelerated this proliferation (6.4 DP) and increased the size of initial clones with no increase in the number of CFU-F. In BM, H and FH, the frequency of CD45− CD14− CD73+ cells in the mononuclear population was 0.02±0.004, 0.47±0.25 and 0.43±0.03%, respectively. That of mesenchymal progenitors (CFU-F) was 15.6±2.7 (BM), 85.18±17.3 (H) and 108.5±7.8 (FH)/10⁶ initial cells, with a majority (60%) of large colonies (>50 cells). The frequency of CFU-F amongst native adherent cells (up to 60%) dramatically decreased since after P0 then appeared relatively stable (down to 7%) during passaging. In parallel, whatever the origin of the MC, the size of colonies decreased progressively, resulting in a majority of <50 cell-colonies at P3. Similarly, we observed that primary culture (P0) showed an average of 15 DP until confluence. After the first trypsinization, cell amplification clearly decreased and remained stable with a mean of 3 DP for each following passage (P1, P2, and P3). Cell proliferation was definitely reduced after about 25 DP, associated with a decrease in colony size.

In conclusion, the majority of native BM adherent cells were mesenchymal progenitors. Their in vitro expansion was associated with a clearly decrease in cloning efficiency amongst their progeny from the earliest culture period and with a gradual decrease in their individual proliferative ability, revealing a marked reduction in NMP proliferative potential in standard culture medium. Expanded cells may not be biologically identical to NMP. Therefore, we confirmed here that most NMP from BM collections were in hematons, tissue aggregates usually eliminated by BM filtration. It is necessary now to define other conditions of culture for NMP amplification.