title: Clonal population of adult stem cells: life span and differentiation potential.
abstract: Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.
authors.affiliation: Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
authors.forename: Mitchel
authors.initials: M
authors.lastname: Seruya
authors.forename: Anup
authors.initials: A
authors.lastname: Shah
authors.forename: Dawn
authors.initials: D
authors.lastname: Pedrotty
authors.forename: Tracey
authors.initials: T
authors.lastname: du Laney
authors.forename: Ryan
authors.initials: R
authors.lastname: Melgiri
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McKee
authors.forename: Henry E
authors.initials: HE
authors.lastname: Young
authors.forename: Laura E
authors.initials: LE
authors.lastname: Niklason
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D024043:Collagen Type II / Q000096:biosynthesis; D003239:Connective Tissue Cells / Q000166:cytology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D017570:MyoD Protein / Q000096:biosynthesis*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D024043:Collagen Type II; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D016212:Transforming Growth Factor beta