title: A Site-Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells.
abstract: The use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) for study and treatment of bone diseases or traumatic bone injuries requires efficient protocols to differentiate hESCs/iPSCs into cells with osteogenic potential and the ability to isolate differentiated osteoblasts for analysis. We have used zinc finger nuclease technology to deliver a construct containing the Col2.3 promoter driving GFPemerald to the AAVS1 site (referred to as a "safe harbor" site), in human embryonic stem cells (H9Zn2.3GFP), with the goal of marking the cells that have become differentiated osteoblasts. In teratomas formed using these cells, we identified green fluorescent protein (GFP)-positive cells specifically associated with in vivo bone formation. We also differentiated the cells into a mesenchymal stem cell population with osteogenic potential and implanted them into a mouse calvarial defect model. We observed GFP-positive cells associated with alizarin complexone-labeled newly formed bone surfaces. The cells were alkaline phosphatase-positive, and immunohistochemistry with human specific bone sialoprotein (BSP) antibody indicates that the GFP-positive cells are also associated with the human BSP-containing matrix, demonstrating that the Col2.3GFP construct marks cells in the osteoblast lineage. Single-cell cloning generated a 100% Col2.3GFP-positive cell population, as demonstrated by fluorescence in situ hybridization using a GFP probe. The karyotype was normal, and pluripotency was demonstrated by Tra1-60 immunostaining, pluripotent low density reverse transcription-polymerase chain reaction array and embryoid body formation. These cells will be useful to develop optimal osteogenic differentiation protocols and to isolate osteoblasts from normal and diseased iPSCs for analysis.
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Xin
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Mary Louise
authors.initials: ML
authors.lastname: Stover
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Shuning
authors.initials: S
authors.lastname: Zhan
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Jianping
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: A Jon
authors.initials: AJ
authors.lastname: Goldberg
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Yongxing
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Liisa
authors.initials: L
authors.lastname: Kuhn
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Reichenberger
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Rowe
authors.affiliation: Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA lichtler@neuron.uchc.edu.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Lichtler
mesh_terms: D000818:Animals; D001857:Bone Matrix / Q000166:cytology; D002454:Cell Differentiation / Q000502:physiology; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D053595:Embryonic Stem Cells / Q000166:cytology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D010006:Osteoblasts / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology; D051381:Rats
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D049452:Green Fluorescent Proteins
keywords: Bone; Gene targeting; Mesenchymal stem cell; Osteoblast; Zinc fingers