naa a22 4500 510739709 CHVBK 20180411083023.0 cr unu---uuuuu 180411e20130401xx s 000 0 eng 10.1007/s12015-013-9434-7 doi (NATIONALLICENCE)springer-10.1007/s12015-013-9434-7 Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures [Elektronische Daten] [Andrei Malko, Maria Villagomez, Jane Aubin, Michal Opas] High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth. Springer Science+Business Media New York, 2013 Micromass culture nationallicence Limb bud nationallicence Chondrogenesis nationallicence Proliferation nationallicence Differentiation nationallicence ALP : Alkaline phosphatase nationallicence BrdU : Bromodeoxyuridine nationallicence BSP : Bone sialoprotein nationallicence Col : Collagen nationallicence FITC : Fluorescein isothiocyanate nationallicence OCN : Osteocalcin nationallicence PBS : Phosphate buffered saline nationallicence PI : Propidium iodide nationallicence PSA : Puck's saline without glucose nationallicence PSG : Puck's saline with glucose nationallicence PTHR1 : Parathyroid hormone related peptide receptor 1 nationallicence SEM : Standard error of the mean nationallicence Malko Andrei Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut Villagomez Maria Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut Aubin Jane Department of Molecular Genetics, University of Toronto, Toronto, Canada aut Opas Michal Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut Stem Cell Reviews and Reports Humana Press Inc 9/2(2013-04-01), 121-131 1550-8943 9:2<121 2013 9 12015 https://doi.org/10.1007/s12015-013-9434-7 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12015-013-9434-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Malko Andrei Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut NATIONALLICENCE 700 1- Villagomez Maria Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut NATIONALLICENCE 700 1- Aubin Jane Department of Molecular Genetics, University of Toronto, Toronto, Canada aut NATIONALLICENCE 700 1- Opas Michal Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada aut NATIONALLICENCE 773 0- Stem Cell Reviews and Reports Humana Press Inc 9/2(2013-04-01), 121-131 1550-8943 9:2<121 2013 9 12015 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer