Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration.

Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-C(Ad)). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-C(Ad) and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r(2) > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

Centrin: its secondary structure in the presence and absence of cations.

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.