title: Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent.
abstract: Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.
authors.affiliation: Orthopaedic and Trauma Surgery Center, University Medical Center Mannheim, Theodor Kutzer Ufer 1-3, 68167, Mannheim, Germany. fickert@uni-heidelberg.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Fickert
authors.forename: Ute
authors.initials: U
authors.lastname: Schröter-Bobsin
authors.forename: Anna-Friederike
authors.initials: AF
authors.lastname: Gross
authors.forename: Ute
authors.initials: U
authors.lastname: Hempel
authors.forename: Claudia
authors.initials: C
authors.lastname: Wojciechowski
authors.forename: Claudia
authors.initials: C
authors.lastname: Rentsch
authors.forename: Denis
authors.initials: D
authors.lastname: Corbeil
authors.forename: Klaus Peter
authors.initials: KP
authors.lastname: Günther
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D000071063:Endoglin; D005260:Female; D005326:Fetal Proteins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D010012:Osteogenesis / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000378:metabolism; D060245:Tetraspanin 29; D018800:Thy-1 Antigens / Q000378:metabolism; D055815:Young Adult; D020410:Activated-Leukocyte Cell Adhesion Molecule
chemical_list: D015720:5'-Nucleotidase; D015703:Antigens, CD; D015816:Cell Adhesion Molecules, Neuronal; D050659:Core Binding Factor Alpha 1 Subunit; D000071063:Endoglin; D005326:Fetal Proteins; D018960:Hyaluronan Receptors; D018799:Intercellular Adhesion Molecule-1; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D060245:Tetraspanin 29; D018800:Thy-1 Antigens; C105613:ALCAM protein, human; D020410:Activated-Leukocyte Cell Adhesion Molecule; C554869:CD9 protein, human; C064206:ENG protein, human
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