SUMMARY: Mesenchymal stem cells are characterized by in vitro high proliferation and multilineage potential maintenance. This study aimed to isolate and characterize equine YS mesenchymal stem cells and compare these with amniotic membranes. The yolk sac (YS) and amniotic membranes (AM) were obtained from 20 pregnant mares with gestational age around 30 days. Cells were cultured in α-MEM supplemented with 15 % FBS, 1 % antibiotic solution, 1 % L-glutamine and 1 % nonessential amino acids. To cell characterization we used cytogenetic analysis, fibroblast colony-forming unit assays, cell growth curves, immunophenotyping, flow cytometry, differentiation assays and teratoma formation. Results: Both cell sources presented fibroblastoid and epithelioid-like format. The YS cells have lower colony formation potential then AM ones, 3 versus 8 colonies per 103 plated cells. However, YS cells grew progressively while AM cells showed steady. Both, the YS and amnion cells immunolabeled for Oct-4, Nanog, SSEA-3, cytokeratin 18, PCNA, and vimentin. In addition, presented mesenchymal, hematopoietic, endothelial and pluripotency markers in flow cytometry. Discussion: Both cell sources presented high plasticity and differed into osteogenic, adipogenic, and chondrogenic lineages, and no tumor formation in nude mice was observed. The results suggest that horse YS may be useful for cell therapy such as amnion-derived cells.

RESUMEN: Las células madre mesenquimales se caracterizan por una alta proliferación in vitro y un mantenimiento potencial de múltiples líneas. Este estudio tuvo como objetivo aislar y caracterizar las células madre mesenquimales del saco vitelino equinas y compararlas con las membranas amnióticas. Se obtuvo el saco vitelino (SV) y las membranas amnióticas (MA) de 20 yeguas preñadas con edad gestacional de aproximadamente 30 días. Las células se cultivaron en α -MEM suplementado con 15 % de FBS, 1 % de solución antibiótica, 1 % de L-glutamina y 1 % de aminoácidos no esenciales. Para la caracterización celular utilizamos análisis citogenéticos, ensayos de unidades de colonias de fibroblastos, curvas de crecimiento celular, inmunofenotipaje, citometría de flujo, ensayos de diferenciación y formación de teratomas. Ambas fuentes celulares presentaron formato fibroblastoideo y epitelioide. Las células SV tienen un potencial de formación de colonias más bajo que las de MA, 3 versus 8 colonias por 103 células en placa. Sin embargo, las células SV crecieron progresivamente mientras que las células MA se mostraron estables. Tanto las células YS como las células amnios están inmunomarcadas para Oct-4, Nanog, SSEA-3, citoqueratina 18, PCNA y vimentina. Además, presentó marcadores mesenquimales, hematopoyéticos, endoteliales y pluripotenciales en citometría de flujo. Ambas fuentes celulares presentaron alta plasticidad y diferían en linajes osteogénicos, adipogénicos y condrogénicos, y no se observó formación de tumores en ratones. Los resultados sugieren que el SV de caballo puede ser útil para la terapia celular, como las células derivadas de amnios.

RESUMO: Modelos animais são essenciais para entender a placenta humana sadia. Neste sentido os roedores relacionados ao porquinho da índia tornaram-se foco para tal entendimento. Em particular, os processos de invasão trofoblástica são semelhantes. O último está associado a uma área especializada, a subplacenta. Uma vez que os resultados anteriores mostraram diferenças entre o porquinho da índia e seu relativo o preá, buscamos estudar o desenvolvimento subplacentário com mais detalhes. Pesquisamos 16 fêmeas gestantes de 14 a 55 dias de gestação por meio de histologia, morfometria, imuno-histoquímica e microscopia eletrônica. A sobreposição entre os sistemas sanguíneos materno e fetal apresentou-se com íntima relação, sugerindo alguns processos de troca. A proliferação foi revelada por três métodos independentes, sendo mais ativos no início e metade da gestação, o que corroborou com os resultados anteriores. Embora a degeneração dos tecidos tenha ocorrido, a subplacenta foi mantida até o termo gestacional com acesso à vascularização fetal, apoiando uma hipótese sobre a liberação de substâncias para a unidade fetal em gestação avançada. Em contraste com outras espécies, o trofoblasto extraplacentário mostrou uma mudança de flâmulas sinciciais para células gigantes durante a metade da gestação. As visualizações sobre a placentação em caviomorfos foram influenciadas pelo porquinho da índia, mas nossos dados apoiaram estudos recentes de que a subplacenta apresentava uma plasticidade muito maior. Em relação ao crescimento subplacentário, a degeneração e provavelmente também os processos de troca, o preá e outras espécies apresentaram um padrão mais basal de caviomorfos do que o porquinho da índia. Tais diferenças devem ser consideradas, ao escolher os modelos animais mais adequados para fins especiais em comparação com a placentação humana.

ABSTRACT: Animal models are essential to understand healthy human placentation. Guinea pig related rodents became on focus for such purposes. In particular, processes of trophoblast invasion are similar. The latter is associated with a specialized area, the subplacenta. Since previous results showed differences between the guinea pig and its close relative Galea spixii, we aimed to study subplacental development with more detail. We investigated 16 pregnant females of 14 to 55 days of gestation by means of histology, morphometrics, immunohistochemistry and electron microscopy. The overlap between the fetomaternal blood systems resulted as intimate, suggesting some exchange processes. Proliferation was revealed by three independent methods, being most active in early and mid-gestation, which was in accordance to former results. Though degeneration of tissues took place, the subplacenta was maintained towards term with access to the fetal vascularization, supporting a hypothesis about the release of substances to the fetal unit in advanced gestation. In contrast to other species, the extraplacental trophoblast showed a shift from syncytial streamers to giant cells during mid-gestation. Views on placentation in caviomorphs were influenced by the guinea pig, but our data supported recent studies that the subplacenta had a much greater placidity. In regard to subplacental grow, degeneration and likely also exchange processes, Galea and other species showed a more basal pattern of caviomorphs than the guinea pig. Such differences should be considered, when choosing most adequate animal models for special purposes in comparison to human placentation.

Hystricognathi represent a monophyletic taxon within Rodentia. Since phylogenetically analyzed morphological systems are essential for revealing evolutionary processes, this study identifies evolutionary character transformations on the stem lineage of Hystricognathi as derived from the author's own work and the literature. Data so far indicate that evolutionary transformations in the rostral head region, the loss of tactile ability in the outer nasal skin and the mobile arrangement of the associated cartilage, were allied with a switch from omnivorous to herbivorous and fiber-rich nutrition. Additional character transformations in the skull assist in digesting such food. Structures associated with reproduction and placentation show a remarkable pro portion of derived character conditions: the chorioallantoic placenta has a ring-shaped organization and growth structure which optimizes the capacity for passive diffusion, a subplacenta occurred as a specialized region responsible for placental invasion and the inverted yolk sac facilitates substance exchange with the main placenta. Finally, precocial newborns evolved as a derived condition within Rodentia. All things considered, a mode of reproduction is indicated, which does not demand excessive additional energy intake by the mother and is in accordance with her low energetic diet. Hystricognathi possess major character transformations that represent prerequisites for their successful radiation at the time when more open ecosystems and grasslands evolved during Earth history. The analysis resulted in the reconstruction of a life-near picture of the hystricognath stem species pattern with high explanatory power in terms of changes in space and time and their interdependence with biodiversity.