Osteoblast differentiation of equine induced pluripotent stem cells

Abstract

Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including COL1A1, SPARC, SPP1, IBSP, RUNX2 and BGALP. We also demonstrate that iPSC-osteoblasts are able to produce a mineralised matrix with both calcium and hydroxyapatite deposition. Alkaline phosphatase activity is also significantly increased during osteoblast differentiation. Although the genetic background of the iPSC donor animal affects the level of differentiation observed after 21 days of differentiation, less variation between lines of iPSCs derived from the same horse was observed. The successful, direct, differentiation of equine iPSCs into osteoblasts may provide a source of cells for future regenerative medicine strategies to improve fracture repair in horses undergoing surgery. iPSC-derived osteoblasts will also provide a potential tool to study equine bone development and disease.

Biocompatible Three-Dimensional Printed Thermoplastic Scaffold for Osteoblast Differentiation of Equine Induced Pluripotent Stem Cells

“…Bone mineralised requires the activity of alkaline phosphatase (ALP) 28 and we demonstrated that over the 21 days of differentiation there is an increase ALP activity. As we reported previously 16 , and similar to human and mouse ESCs 29 , undifferentiated equine iPSCs express a low level of ALP. ALP activity is increased approximately 20.5 fold after 21 days of differentiation in 3D.…”

“…We have previously reported the successful 2D differentiation of equine iPSCs into osteoblasts 16 and here we report the use of a novel, thermoplastic 3D printed scaffold to allow in vitro 3D osteoblast differentiation of equine iPSCs.…”

“…They were modified to constitutively express GFP using retroviral integration. Briefly, phoenix-gag-pol (PGP) cells were transfected with Six lines of previously derived equine iPSCs 15,16 from three different horses were cultured on mitotically inactivated mouse embryonic fibroblasts in DMEM/F12, supplemented with: 15% FCS, 2 mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all Invitrogen), 1000 U/ml leukaemia inhibitory factor (LIF, Sigma), and 10 ng/ml basic fibroblast growth factor (bFGF, Peprotech, NJ, USA). Colonies were passaged mechanically every 5 to 7 days in the presence of 2 μM Thiazovivin (StemGent, Cambridge, MA).…”

“…However, we were unable to find a successful method for extracting live cells from the scaffold to determine actual cell numbers at the end of the 21 days of differentiation. The extraction of live cells is likely complicated by the fact that upon differentiation into osteoblasts the cells produce a mineralised matrix 16 .…”

“…Induced pluripotent stem cells are cells that are derived from somatic adult cells and have been reprogrammed such that they resemble an embryonic-like state and are capable of indefinite proliferation and can form cells from all three germ line lineages (endoderm, ectoderm and mesoderm) 11 including bone-forming osteoblasts [12][13][14] . We have successfully generated iPSC lines from equines 15 and have developed methods to differentiate the iPSCs into osteoblasts using traditional 2D cell culture techniques 16 .…”