Developmental potential of somatic stem cells in mammalian adults.

Traditionally, somatic tissue-derived stem cells of mammalian adults have been viewed as pluripotent precursors capable of lifelong maintenance of cellular compartments typical of the tissue in which they reside. However in recent years, in vitro cultures and in vivo transplantation assays have indicated that adult somatic stem cell of various species are capable of adopting multiple fates. Bone marrow cells can give rise to a wide array of phenotypes, including blood, endothelial, bone, cartilage, fat, tendon, lung, liver, muscle, marrow stroma, and even brain cells. Conversely, neural stem cells as well as progenitors present in the muscle may contribute to blood cell production, indicating that adult stem cells present in numerous tissues may generate multiple cell types even of different dermal origin. Therefore, the developmental potential of adult somatic stem cells might be reassessed, although the mechanisms that ultimately lead to determination of cell fate are not completely defined. The successful long-term culturing and expansion of somatic adult stem cells together with their intrinsic versatility leads to future hope of stem cell therapeutic use in a wide spectrum of diseases and disorders of several, even not easily accessible, tissues.

Human GM-CSF interaction with the alpha-chain of its receptor studied using surface plasmon resonance.

A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) with genetically engineered alpha-chain subunits of its specific receptor (GM-Ralpha). Western blot analysis of GM-Ralpha confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Ralpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Ralpha subsequently injected over the sensing layer. GM-Ralpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Ralpha with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Ralpha with dilutions of neutralizing anti-GM-Ralpha antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CSF over the GM-CSF matrix, before injecting GM-Ralpha. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Ralpha. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.