title: A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.
abstract: In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ochiai-Shino
authors.affiliation: Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kato
authors.affiliation: Department of Ultrastructural Science, Tokyo Dental College, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sawada
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Onodera
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Saito
authors.affiliation: Department of Oral and Maxillofacial Surgery, Division of Tissue Engineering, Faculty of Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takato
authors.affiliation: Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, Tokyo, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Shibahara
authors.affiliation: Department of Endodontics and Clinical Cariology, Tokyo Dental College, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Muramatsu
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Azuma
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D016130:Immunophenotyping; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D010012:Osteogenesis; D000091042:Reelin Protein
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers; D000091042:Reelin Protein; D000469:Alkaline Phosphatase; C000717127:RELN protein, human