Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P