Abstract

Equine pluripotent stem cells have been derived from the inner cell mass of blastocyst stage embryos (embryonic stem cells, ESCs) and from the reprogramming of adult cells into a pluripotent state (induced pluripotent stem cells, iPSCs).

ESCs differentiate into tenocytes in vivo following their injection into an injured horse tendon and in vitro in response to transforming growth factor beta 3 (TGFβ3) and/or 3D culture in an anchored collagen matrix.

Here we demonstrate that while equine iPSCs express tendon-associated genes and proteins following TGF -β3 exposure in 2D culture, they cannot remodel a 3D collagen matrix to generate “artificial tendons”. This demonstrates the utility of the 3D in vitro tendon assay for measuring functional tendon differentiation and the need for more detailed studies to be performed to determine if epigenetic differences exist between equine iPSCs and ESCs.

To ensure the safety of pluripotent stem cells in the clinic the mechanisms controlling their differentiation must be understood. We have demonstrated that the transcription factor scleraxis is required for tendon differentiation by equine ESCs, as the overexpression of a short hairpin RNA (shRNA) to scleraxis leads to a failure to generate artificial tendons in 3D culture. Scleraxis is also required for the generation of artificial tendons by fetal tenocytes and its knockdown using shRNA results in a significant reduction in the expression of COL1A1 , COMP and SOX9 . These effects can subsequently be rescued through scleraxis overexpression. In contrast, adult tenocytes expressing a shRNA to scleraxis demonstrated no changes in the expression levels of the genes measured and the cells continued to generate artificial tendons as normal in 3D culture.

A further safety consideration is in understanding the immune properties of pluripotent stem cells and their differentiated derivatives. We have demonstrated that allogeneic ESCs do not produce a detectable immune response following their injection into the injured tendon in a small scale in vivo study. Furthermore, undifferentiated ESCs and spontaneously differentiated ESCs do not stimulate the proliferation of co-cultured allogeneic peripheral blood mononuclear cells in vitro. However, more detailed studies on the immune properties of ESC -derived tendon cells and the effect of an inflammatory environment are required.