In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell‐Derived Osteoprogenitors

Abstract

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

Stem cells applications in bone and tooth repair and regeneration: New insights, tools, and hopes

“…They have also shown that the impairment of bone regeneration is probably due to a damage of the surrounding skeletal muscle (reviewed in Owston, Giannoudis, & Jones, ). For example, a recent study from Cao's laboratory (Islam et al, ) has shown that fluorescently labeled human embryonic stem cells‐derived osteoprogenitors (hESC‐OPs) are able to maintain both their GFP expression for the long term and their potential for osteogenic differentiation in culture. This will help in the future application of these fluorescently labeled hESC‐OPs in the noninvasive assessment of bone regeneration and therapeutic efficacy.…”

Light-Controlled BMSC Sheet–Implant Complexes with Improved Osteogenesis via an LRP5/β-Catenin/Runx2 Regulatory Loop

“…Cell tracking has been a focus of active research in regenerative engineering. , Enhanced green fluorescent protein (EGFP) reporter gene, , luciferase reporter gene, − and 5-bromodeoxyuridine , are often used to image engineered cells. The luciferase and EGFP reporter genes, which have been used extensively in tumor cells, are rarely employed in implantology.…”

A hierarchical vascularized engineered bone inspired by intramembranous ossification for mandibular regeneration

“…The green fluorescent protein (GFP)-MSCs/GFP-ECs differentiated from GFP-hESCs as reported 53 were cultured in PG/PGM and observed using an inverted laser scanning confocal microscope (Leica, Germany) at days 1, 3, 5, and 7. Serial optical sections were captured and reconstructed into 3D images, and the cell volume and sphericity were calculated using the Imaris 9.0.1 software (Bitplane, Switzerland).…”