title: Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection.
abstract: Mesenchymal stem cells (MSCs) are typically enriched from bone marrow via isolation of the plastic adherent, fibroblastoid cell fraction. However, plastic adherent cultures elaborated from murine bone marrow are an admixture of fibroblastoid and hematopoietic cell types. Here we report a reliable method based on immunodepletion to fractionate fibroblastoid cells from hematopoietic cells within plastic adherent murine marrow cultures. The immunodepleted cells expressed the antigens Sca-1, CD29, CD44, CD81, CD106, and the stem cell marker nucleostemin (NST) but not CD11b, CD31, CD34, CD45, CD48, CD90, CD117, CD135, or the transcription factor Oct-4. They were also capable of differentiating into adipocytes, chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo. Therefore, immunodepletion yields a cell population devoid of hematopoietic and endothelial cells that is phenotypically and functionally equivalent to MSCs. The immunodepleted cells exhibited a population doubling time of approximately 5-7 days in culture. Poor growth was due to the dramatic down regulation of many genes involved in cell proliferation and cell cycle progression as a result of immunodepletion. Exposure of immunodepleted cells to fibroblast growth factor 2 (FGF2) but not insulin-like growth factor (IGF), murine stem cell factor, or leukemia inhibitory factor (LIF) significantly increased their growth rate. Moreover, 82% of the transcripts down regulated by immunodepletion remain unaltered in the presence of FGF2. Exposure to the later also reversibly inhibited the ability of the immunodepleted cells to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Therefore, FGF2 appears to function as a mitogen and self-maintenance factor for murine MSCs enriched from bone marrow by negative selection.
authors.affiliation: Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
authors.forename: Melody
authors.initials: M
authors.lastname: Baddoo
authors.forename: Katy
authors.initials: K
authors.lastname: Hill
authors.forename: Robin
authors.initials: R
authors.lastname: Wilkinson
authors.forename: Dina
authors.initials: D
authors.lastname: Gaupp
authors.forename: Catherine
authors.initials: C
authors.lastname: Hughes
authors.forename: Gene C
authors.initials: GC
authors.lastname: Kopen
authors.forename: Donald G
authors.initials: DG
authors.lastname: Phinney
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D017931:DNA Primers; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D015870:Gene Expression; D006133:Growth Substances / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010969:Plastics; D016133:Polymerase Chain Reaction
chemical_list: D015703:Antigens, CD; D017931:DNA Primers; D006133:Growth Substances; D010969:Plastics; D005346:Fibroblast Growth Factors