295 EQUINE AMNION-DERIVED MESENCHYMAL STEM CELLS: POSSIBLE IMPLICATION IN ENDOMETRIAL REGENERATION

Abstract

In veterinary medicine, as in human, amnion is an attractive source of mesenchymal stem cells (MSC), which poses no ethical dilemmas, allows highly efficient recovery of cells without the requirement of invasive procedures and shows immunomodulatory properties. We previously demonstrated that equine amniotic membrane-derived cells (AMC) not only exhibit specific stem cell properties with respect to expression of pluripotent (OCT-4, TRA-1-60, and SSEA-4) and adult (CD44, CD105, CD29, and CD166) stem cell markers but also possess differentiation potential in vitro and the capability to regenerate tendons in vivo after spontaneous lesions when allogeneically transplanted. Moreover, we reported evidence that at the first passages (P) in culture (until P5), AMC express MHC-class I but not MHC-class II and are well tolerated in vivo. In the present study, we further characterised AMC in vitro in order to evaluate their potential application in the treatment of endometritis in vivo. In particular, the amniotic membrane in toto and AMC have been compared to the endometrial tissue in toto and to cells isolated from endometrium for the expression of genes involved in the proliferation and differentiation of uterine MSC during early pregnancy (AbdB-like Hoxa genes) and those influencing preimplantation conceptus development (progesterone receptor, PR, and oestrogen receptors ERα and ERβ). The AMC were isolated as recently reported by Lange-Consiglio et al. (2011 Open J. Tissue Eng. Regen. Med. 4), and endometrial cells were obtained according to the protocol described by Donofrio et al. (2008 Reprod. Biol. Endocrinol. 6, 65) for bovine cells, and slightly modified for equine cells. Total RNA was extracted from both tissues and from AMC and endometrium-derived cells immediately after isolation (P0). Reverse transcription-PCR was performed according to the standard procedures, using GAPDH and HPRT1 as reference genes. Expression of HOXA9 and PR was confirmed in all samples examined, whereas mRNA for ERβ was only detected in endometrial tissue and in cells derived from it. Expression of ERα was observed only in endometrial tissue. The expression of genes crucially involved in patterning of the female reproductive tract (HOXA9 and PR) in amnion and cells derived from it suggests that this source shares similar molecular properties with endometrium. Further studies are required to explore uterine mesenchymal-like features shared by AMC (i.e. verifying the expression of Wnt7α, Wnt5α, and Wnt4α, or the presence of the more recently characterised membrane-bound intracellular progesterone receptors PGRMC1 and mPR). These preliminary results provide an intriguing indication of the possible implication of amnion-derived cells in endometrial regeneration, in particular when poor endometrial proliferation is associated with infertility or poor pregnancy outcome.