Browsing by Author "Cortez, Jahaira"

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    Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids
    (2024) Cortez, Jahaira; Torres, Cristian G.; Parraguez, Victor H.; De los Reyes, Monica; Peralta, Oscar A.
    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 x 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 < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.
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    Potential of mesenchymal stromal/stem cells and spermatogonial stem cells for survival and colonization in bull recipient testes after allogenic transplantation
    (2024) Segunda, Moises N.; Cortez, Jahaira; Diaz, Carlos; Arancibia, Richard; Torres, Cristian G.; Parraguez, Victor H.; de los Reyes, Monica; Peralta, Oscar A.
    Stem cell transplantation into seminiferous tubules of recipient testis could become a tool for fertility restoration, genetic improvement, or conservation of endangered species. Spermatogonial stem cells (SSCs) are primary candidates for transplantation; however, limited abundance, complexity for isolation and culture, and lack of specific markers have limited their use. Mesenchymal stromal/stem cells (MSCs) are multipotent progenitors that are simple to isolate and culture and possess specific markers for identification, and immune evasive and migratory capacities. The objective of the present study was to evaluate the potential for survival and colonization in seminiferous tubules of two different concentrations of bovine fetal adipose tissue-derived MSCs (ATMSCs), native of pre-induced, and to compare the fate of bovine adult peripheral blood-derived MSCs (PB-MSCs) and SSCs after allogenic transplantation in testis of recipient bulls. In experiment 1, AT-MSCs at two concentrations (1x107 and 2x107; n = 3) or pre-exposed to 2 mu M testosterone and 1 mu M retinoic acid (RA) for 14 days (n = 5) were evaluated. In experiment 2, adult PB-MSCs and SSCs (4x107 cells each) pre-exposed to Sertoli cell conditioned media (SCs/CM; n = 4) for 14 days were compared. Each cell type was separately labelled with PKH26 and then transplanted into testes of 8-month-old recipient bulls. Four weeks (Exp. 1) and two weeks (Exp. 2) after transplantation, testicular tissue was processed for confocal microscopy detection of PKH26-positive cells. Mean number of PKH26-positive cells were higher (P < 0.05) in testis transplanted with 2x107 AT-MSCs in the proximal (6.7 f 3.7) and medial (6.6 f 3.2) sections compared to testis transplanted with 1x107 ATMSCs (proximal: 1.9 f 1; medial: 1.9 f 1) sections or pre-induced AT-MSCs (proximal: 4.7 f 5.6; medial: 3.8 f 4.1). In Exp. 2, mean number of PKH26-positive SSCs in medial testicular section (22.5 f 1.3) were higher (P <0.05) compared to respective section in PB-MSCs group (17 f 4.2). Thus, in vivo data indicates that a higher number of transplanted AT-MSCs resulted in more cells surviving and colonizing seminiferous tubules; however, pre-induction with testosterone and RA did not improve these capacities. SSCs displayed a greater capacity for survival and colonization in recipient seminiferous tubules; however, PB-MSCs were observed in all sections of testis after two weeks of transplantation.