title: Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages.
abstract: We recently developed a simple strategy for the enrichment of mesenchymal stem cells (MSCs) with the capacity for osteoblast, chondrocyte, and adipocyte differentiation. On transplantation, the progenitor-enriched fraction can regenerate bone with multiple lineages of donor origin. Although comprising multiple precursor cell types, the population is enriched >100-fold in osteoprogenitors, hence the name "highly purified osteoprogenitors" (HipOPs). To establish a new modified method of purifying pure MSCs, it is useful to know the expression patterns of surface markers on heterogeneous MSCs and committed cells such as osteoblasts, adipocytes, and chondrocytes. However, calcium deposition by osteoblasts is a critical obstacle in visualizing the expression patterns of surface markers. We now report a new method of separating differentiated osteoblastic HipOPs (OB-HipOPs) from calcium deposits using the Percoll density gradient centrifugation technique. After centrifuge separation, calcium deposits were observed at the bottom of the centrifuge tube, and living OB-HipOPs were harvested from the 10-70% fractions. However, there were no living cells in the 70-80% fraction. We concluded that living OB-HipOPs are separated by one 10-70% Percoll gradient. Furthermore, we analyzed the expression patterns of putative MSC markers on differentiated HipOPs. FACS analysis revealed that Sca-1, CD44, CD73, CD105, and CD106 were decreased in OB-HipOPs. In adipogenic- and chondrogenic-HipOPs, Sca-1, CD73, CD105, and CD106 were decreased. This new technique is a helpful tool to identify MSC surface markers and to clarify in more detail the differentiation stages of osteoblasts.
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Yamamoto
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Shousaku
authors.initials: S
authors.lastname: Itoh
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Yamauchi
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Matsushita
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Ikeda
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Haruna
authors.initials: H
authors.lastname: Naruse
authors.affiliation: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Hayashi
mesh_terms: D017667:Adipocytes / Q000166:cytology; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D019902:Chondrocytes / Q000166:cytology; D059630:Mesenchymal Stem Cells; D051379:Mice; D010006:Osteoblasts / Q000166:cytology; D010012:Osteogenesis
keywords: DIFFERENTIATION; METHODOLOGY; OSTEOBLAST; PERCOLL GRADIENT; SURFACE MARKER