Abstract
Summary Statement
Low-intensity pulsed ultrasound (LIPUS) enhanced osteogenic differentiation of osteoprogenitor cells derived from mouse induced pluripotent cells (iPSCs) without embryoid body formation. Our findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs.
Introduction
iPSCs represent a promising cell source for regenerative medicine such as bone regeneration because of their unlimited self-renewal property and ability of differentiation into all somatic cell types. Recently, we developed an efficient protocol for generating a highly homogeneous population of osteoprogenitor cells from embryonic stem cells by using a direct-plating method without EB formation step. It is well-recognised that LIPUS accelerates the fracture healing. There have been several reports showing that LIPUS stimulates the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. To date, effect of LIPUS on iPSCs remains unknown. In this study, we investigated in vitro effect of LIPUS on osteogenic differentiation of osteoprogenitor cells derived from mouse iPS cells via a direct-plating method.
Methods
Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates without feeders in MSC medium and FGF-2. Adherent fibroblastic cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. For osteogenic differentiation, DPCs were divided into two groups: (1) control group: DPCs cultured in osteogenic medium (OM) without LIPUS, and (2) LIPUS group: DPCs cultured in OM with LIPUS treatment. LIPUS was given through the bottom of the culture plates for 20 minutes daily. After 14-day culture, osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin red S staining. Expression of osteoblast-related genes, Rnux2 and ALP was also analyzed by real-time PCR.
Results
Flow cytometry analysis revealed DPCs had similar characteristics to MSCs. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs, suggesting DPCs lost pluripotency. After 14-day osteogenic induction, ALP activity was shown to be higher in LIPUS group than control group on days 3 and 7. Real-time PCR analysis revealed that in LIPUS group, expression level of Runx2 on day 1 and that of ALP on days 3 and 5 were significantly up-regulated compared to control group. The quantity of calcium deposition measured by Alizarin red staining on day 14 was shown to be higher in LIPUS group than control group.
Conclusion
The novel direct-plating method described here provides a significant technical advance over conventional methods of isolating iPSC-induced osteoprogenitor cells by avoiding the embryonic body formation that often leads to heterogeneous, variable, and unpredictable osteogenic differentiation. Our results demonstrated that osteogenic differentiation of osteoprogenitor cells from iPSCs was robustly increased by LIPUS treatment. LIPUS may be a promising enhancer of osteogenesis of iPSCs. These findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs.
