The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures obtained in monolayer differentiation had a big nucleus and a surrounding ZP4 coat, the unique attributes of a female gamete. These oocyte-like structures, in extended cultures, showed embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence. This, as per our knowledge, is first such study in higher mammals, especially farm animals, and assumes significance for its potential use in transgenic animal production, elite animal conservation and propagation, augmentation of reproductive performance in poor breeding buffalo species, and as a model for understanding human germ cell formation.