Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation

Takefumi Sone,Hideyuki Okano,Satoshi Ohte,Takehito Ouchi,Kohnosuke Mitani,Hiroki Sasanuma,Arei Miyamoto,Mahito Nakanishi,Takenobu Katagiri,Sho Tsukamoto,Masashi Shin

doi:10.1038/s41598-019-46105-y

Abstract

We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

Highlights

Osteoblasts are specialized cells for bone formation and are derived from undifferentiated mesenchymal progenitor cells
We showed the dual usage of a novel adenoviral vector, HDAd-hOC-Venus, which allows for both the generation of knock-in reporter cell lines by homologous recombination in human induced pluripotent stem cells (iPSCs) and postnatal visualization and separation of bone-forming mature osteoblasts by transient infection into mouse and human cells
Our helper-dependent adenoviral vector (HDAdV)-based system allows for the transient expression of Venus without chromosomal integration in the target cells

Summary

Introduction

Osteoblasts are specialized cells for bone formation and are derived from undifferentiated mesenchymal progenitor cells. It is a less-toxic and transient expression vector of Venus both in vitro and in vivo in mature osteoblasts of various species[10,11,12] It can be used as an efficient site-specific gene-targeting vector through homologous recombination in order to establish reporter knock-in (KI) cell lines[13,14,15,16] without introducing DNA double-strand breaks on and off target sites using programmable nucleases[17]. Using this system, we established a KI human iPSC line to monitor osteogenic differentiation and isolated living mature osteoblasts by fluorescence-activated cell sorting (FACS) from mixed murine POB cultures

Methods

Results

Conclusion