Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells.
Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1-/ALP+ and STRO-1-/ALP- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA-binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1+/ALP- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1+/ALP- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.
Gronthos
S
S
Matthew Roberts Laboratory, Leukemia Research Unit, Hanson Center for Cancer Research, I.M.V.S., Adelaide, South Australia, Australia.
Zannettino
A C
AC
Graves
S E
SE
Ohta
S
S
Hay
S J
SJ
Simmons
P J
PJ
D000368:Aged; D000369:Aged, 80 and over; D000469:Alkaline Phosphatase / Q000276:immunology*; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000096:biosynthesis*; D015415:Biomarkers; D015519:Bone Density / Q000502:physiology; D001846:Bone Development / Q000502:physiology*; D001857:Bone Matrix / Q000166:cytology / Q000254:growth & development / Q000276:immunology; D019070:Cell Lineage; D002478:Cells, Cultured; D006801:Humans; D008875:Middle Aged; D010012:Osteogenesis / Q000502:physiology; D010641:Phenotype; D012016:Reference Values; D014157:Transcription Factors / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015415:Biomarkers; D014157:Transcription Factors; D000469:Alkaline Phosphatase