Cell Identity, Proliferation, and Cytogenetic Assessment of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

The aim of the present work was to determine proliferation capacity, immunophenotype and genome integrity of mesenchymal stromal cells (MSCs) from horse umbilical cord blood (UCB) at passage stage 5 and 10. Passage 4 cryopreserved UCB-MSCs from six unrelated donors were evaluated. Immunophenotypic analysis of UCB-MSC revealed a cell identity consistent with equine MSC phenotype by high expression of CD90, CD44, CD29, and very low expression of CD4, CD11a/18, CD73, and MHC class I and II antigens. Proliferative differences were noted among the UCB-MSC cultures. UCB-MSCs karyotype characteristics at passage 5 (eg, 2n = 64; XY, or XX) included 20% polyploidy and 62% aneuploidy. At passage 10, the proportion of polyploidy and aneuploidy was 21% and 82%, respectively, with the increase in aneuploidy being significant compared with passage 5. Furthermore, conventional GTG-banded karyotyping revealed several structural chromosome abnormalities at both passage 5 and 10. The clinical relevance of such chromosome instability is unknown, but determination of MSC cytogenetic status and monitoring of patient response to MSC therapies would help address this question.

An atomic model of the thin filament in the relaxed and Ca2+-activated states.

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering.

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.