Open AccessA Site‐Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells
Author(s) -
Xin Xiaonan,
Jiang Xi,
Wang Liping,
Stover Mary Louise,
Zhan Shuning,
Huang Jianping,
Goldberg A. Jon,
Liu Yongxing,
Kuhn Liisa,
Reichenberger Ernst J.,
Rowe David W.,
Lichtler Alexander C.
Publication year - 2014
Publication title -
stem cells translational medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.781
H-Index - 71
eISSN - 2157-6580
pISSN - 2157-6564
DOI - 10.5966/sctm.2013-0128
Subject(s) - embryonic stem cell , embryoid body , induced pluripotent stem cell , stem cell , microbiology and biotechnology , biology , population , alkaline phosphatase , chemistry , cellular differentiation , mesenchymal stem cell , genetics , biochemistry , medicine , environmental health , gene , enzyme
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.