Induced Pluripotent Stem Cells from Animal Models: Applications on Translational Research

Over the history of humankind, knowledge acquisition regarding the human body, health, and the development of new biomedical techniques have run through some animal model at some level. The mouse model has been primarily used as the role model for a long time; however, it is severely hampered regarding its feasibility for translational outcomes, in particular, to preclinical and clinical studies. Herein we aim to discuss how induced pluripotent stem cells generated from non-human primates, pigs and dogs, all well-known as adequate large biomedical models, associated or not with gene editing tools, can be used as models on in vivo or in vitro translational research, specifically on regenerative medicine, drug screening, and stem cell therapy

Reproductive biology of male viscacha (Lagostomus maximus)-Partnership between Brazil and Argentina for the integration of biotechnology in breeding of endangered animals

Atualmente muitos são os estudos com animais silvestres, no entanto, pouco se sabe sobre os aspectos biológicos dos machos. Os Histricomorpha são uma subordem da ordem Rodentia, que representa 43% das 4629 espécies reconhecidas encontradas na classe Mammalia. Os roedores são animais que oferecem uma extraordinária variedade de adaptações ecológicas, suportando os mais diversos tipos de climas e altitudes, podendo com isso apresentar grande diversidade funcional, a viscacha é um roedor pertencente a esta subordem, e de grande importância para o ambiente em que habitam, pois, representam a base da cadeia alimentar. Por esta razão, esta pesquisa ter por objetivo descrever morfologicamente o aparelho reprodutor do macho de viscacha. Foram utilizados seis macho adultos de viscacha, realizou-se análise macro e de microscópia de luz, eletrônica e imunohistoquímicas do sistema reprodutor masculino, bem como a espermatogênese deste animal. Através das análises realizadas, a viscacha apresenta o aparelho reprodutor composto por um par de testículos, epidídimo e ducto deferente e as seguintes glândulas genitais acessórias: próstata, vesícula seminal e glândula bulbouretral. Estas estruturas são semelhantes às de outros roedores já descritos, como a Chinchila e Cutia. A análise espermatogênica demonstrou que esta exibe alterações significativas de seus parâmetros, em sincronia com o ambiente externo e o ciclo reprodutivo. Conclui-se, com os animais estudados, que a morfologia do aparelho reprodutor do L.maximus viscacha é semelhante aos demais roedores pertencentes à subordem dos Histricomorfos.Many groups are focused their studies on wild animal reproductive biology. However little is still known about the male biology. The Histricomorpha is a suborder of the Rodentia order, which represents 43% of 4,629 recognized species found in the class of Mammalia. Rodents are animals that offer an extraordinary variety of ecological adaptations, supporting all kinds of climates and altitudes, and this may have vast functional diversity. The viscacha is a rodent animal that belongs to this suborder, and it has a great importance to the environment it inhabits, since they represent the base of the food chain. For this reason, this research aims to describe the morphology of the reproductive system of male viscacha. Six adult viscacha males were used for gross anatomy analysis, for light and electron microscopic analysis, and immunhistochemical analysis to describe the normal histology and spermatogenesis of the reproductive tract of adult male viscacha. The reproductive system of the male viscacha consists in a pair of testicles, epididymis and vas deferens and accessory genital glands following: prostate, seminal vesicles and bulbourethral gland. These structures are similar to those already described for other rodents such as Chinchilla and Agouti. The analysis of spermatogenesis suggested that the male viscacha is able to synchronize its spermatogenesis according to the environment, which the animal is inserted in, and its reproductive cycle. This study showed that the reproductive system of the viscacha (L.maximus) is morphologically similar to other rodents belonging to the suborder histricomorphas

Spermatogenesis xenogeneic after stem cell transplantation in canine testis of mice

As células tronco espermatogoniais (SSCs) são caracterizadas pela capacidade de autorrenovação, proliferação e transmissão das informações genéticas. Em caninos a primeira tentativa de xenotransplante não obteve o sucesso da produção de espermatozoides, no entanto, há evidências de que as células testiculares xenogênicas podem ser transplantadas no testículo do animal hospedeiro, e gerar espermatozoides viáveis do doador. Portanto, este estudo tem como objetivo realizar o xenotransplante das células germinativas caninas em camundongos imunosuprimidos, e com isto promover à produção de espermatozoides caninos viáveis, geneticamente modificados. E por meio desta técnica, analisar a eficiência da espermatogênese pós-transplante. Células germinativas testiculares foram caracterizadas, isoladas e cultivadas de cães pré-púberes, por meio de sistemas de cultura de enriquecimento e fatores de crescimento. As células foram transduzidas com um gene repórter GFP e LacZ, e por um vetor lentiviral para indentificar as SSCs nos testículos receptores. As SSCs transduzidas foram transplantadas nos testículos de camundongos (C57BL/6) tratados com Busulfan, após diferentes períodos os animais receptores foram eutanasiados e analisados. Aos 10 dias de cultivo as células germinativas adultas foram positivas para CD49f, CD117, e com 5 dias uma expressão semelhante de GFRA1 e DAZL, demonstrando a presença de SSCs e algumas células em meiose. Transplantamos 105 células e 20-43% das células transplantadas foram identificadas na membrana basal dos túbulos seminíferos do animal receptor. Portanto, o transplante das células germinativas caninas, mostrou que a purificação e o cultivo realizados são possíveis para obter SSCs caninas, as quais colonizaram os túbulos seminíferos dos camundongos imunodeficientes e mantiveram-se vivas na membrana basal por 90 dias após transplante, mesmo que estes animais tenham distância filogenéticaThe spermatogonial stem cells (SSCs) are characterized by the capacity for self-renewal, proliferation and transmission of genetic information. In canines the first attempt to xenotransplantation did not achieve the success of sperm production. However, there is evidence that testicular xenogeneic cells can be transplanted into the testis of the host animal, and generate viable sperm donor. Therefore, this study aims conduct xenotransplantation of the canine germ cells in immunosuppressed mice, and thereby promote the production of viable sperm canines, genetically modified. And by this technique, analyze the efficiency of post-transplant spermatogenesis. Testicular germ cells were identified, isolated and cultured prepuberes dogs through enrichment culture systems and growth factors. Cells were modificated with a reporter gene GFP and LacZ. The SSCs canine was transplanted in mice (C57Bl/6), after different period and then the recipient animals were euthanized and analyzed. After 10 days in culture the germ cells were positive for CD49f, CD117, and 5 days a similar expression of GFRA1 and DAZL was observed, demonstrating the presence of SSCs and some cells in meiosis. 105 cells were transplanted and 20-43% of the transplanted cells were identified in the basement membrane of the seminiferous tubules after 90 days after transplantation. Therefore, the transplantation of canine germ cells showed that the cultivation and purification are performed possible for canine SSCs, it can colonize the seminiferous tubules the mice infertility remained alive in the basement membrane for 90 days after transplanting, even though these animals have phylogenetic distanc

Step by Step about Germ Cells Development in Canine

Primordial germ cells (PGCs) have been described as precursors of gametes and provide a connection within generations, passing on the genome to the next generation. Failures in the formation of gametes/germ cells can compromise the maintenance and conservation of species. Most of the studies with PGCs have been carried out in mice, but this species is not always the best study model when transposing this knowledge to humans. Domestic animals, such as canines (canine), have become a valuable translational research model for stem cells and therapy. Furthermore, the study of canine germ cells opens new avenues for veterinary reproduction. In this review, the objective is to provide a comprehensive overview of the current knowledge on canine germ cells. The aspects of canine development and germ cells have been discussed since the origin, specifications, and development of spermatogonial canine were first discussed. Additionally, we discussed and explored some in vitro aspects of canine reproduction with germ cells, such as embryonic germ cells and spermatogonial stem cells

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro

Emerging Contributions of Pluripotent Stem Cells to Reproductive Technologies in Veterinary Medicine

The generation of mature gametes and competent embryos in vitro from pluripotent stem cells has been successfully achieved in a few species, mainly in mice, with recent advances in humans and scarce preliminary reports in other domestic species. These biotechnologies are very attractive as they facilitate the understanding of developmental mechanisms and stages that are generally inaccessible during early embryogenesis, thus enabling advanced reproductive technologies and contributing to the generation of animals of high genetic merit in a short period. Studies on the production of in vitro embryos in pigs and cattle are currently used as study models for humans since they present more similar characteristics when compared to rodents in both the initial embryo development and adult life. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine

Evaluation of targeted oxidative stress induced by oxygen-ozone in vitro after ischemic induction

Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans, the second leading cause of death and physical and cognitive disabilities, in developing countries. It presents as an ischemic (more common) or hemorrhagic form. Ozone therapy has been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. Neuroblastoma lineage cells (SH-SY5Y) and canine amniotic membrane stem cells were subjected to 24 hours of hypoxia in an incubator culture chamber. The cells were evaluated by MTT assay, colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. Treatment with low concentrations of ozone (2–10 μg/mL), indicated a possible neuroregenerative effect at low concentrations, correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 μg/mL) promoted an increase in rate of apoptosis and cell death. We developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group