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.

Porcine Germ Cells Phenotype during Embryonic and Adult Development.

Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.

3D culture of mesenchymal stem cells from the yolk sac to generate intestinal organoid.

Organoids are in vitro models that originated from the three-dimensional culture of stem cells with the ability to reproduce part of the in vivo structural and functional specificities of body organs. Intestinal organoids have great relevance in cell therapy, as they provide more accurate information about tissue composition and architecture in relation to two-dimensional culture, in addition to serving as a study model for host interaction and drug testing. The yolk sac (YS) is a promising source of mesenchymal stem cells (MSCs), which are multipotent stem cells with self-renewal ability and potential to differentiated into mesenchymal lineages.

Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state.

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.

induced pluripotency from urine-derived cells in porcine.

Background: The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term and offspring analysis; therefore, suitable for both medicine and animal production.

Aim: To reprogramme into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine.

Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients.

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification.

Isolation and characterization of neural stem cells from fetal canine spinal cord.

Neurogenesis in adult mammals occurs mainly in the subventricular and subgranular areas of the brain, but there are also reports of its occurrence in the spinal cord. In a study on rats, neural stem cells and neuroprogenitor cells could be obtained through primary spinal cord culture, but there are no studies on these cells in canine species, to date. Dogs represent an appropriate animal model for studies on neurogenesis and neurological disorders.

Actions and Roles of FSH in Germinative Cells.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR).

Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation.

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration).

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs).

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models used in pre-clinical research, such as rodents, are not able to meet the criteria to translate the findings to the clinic. Non-human primates, canines, and porcine are considered more adequate models for biomedical research and drug development purposes, mainly due to their physiological, genetic, and anatomical similarities to humans. The swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing.

Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions.

Culture conditions regulate the process of pluripotency acquisition and self-renewal. This study aimed to analyse the influence of the in vitro environment on the induction of porcine induced pluripotent stem cell (piPSCs) differentiation into primordial germ cell-like cells (pPGCLCs). piPSC culture with different supplementation strategies (LIF, bFGF, or LIF plus bFGF) promoted heterogeneous phenotypic profiles.

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.

In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.

The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as culture conditions (e.g., environmental oxygen concentration), and the aging process.

Generation of neural progenitor cells from porcine-induced pluripotent stem cells.

In this study, porcine embryonic fibroblasts (pEFs) were reprogrammed into porcine-induced pluripotent stem cells (piPSCs) using either human or mouse specific sequences for the OCT4, SOX2, c-Myc, and KLF4 transcription factors. In total, three pEFs lines were reprogrammed, cultured for at least 15 passages, and characterized regarding their pluripotency status (alkaline phosphatase expression, embryoid body formation, expression of exogenous and endogenous genes, and immunofluorescence). Two piPSC lines were further differentiated, using chemical inhibitors, into putative neural progenitor-like (NPC-like) cells with subsequent analyses of their morphology and expression of neural markers such as NESTIN and GFAP as well as immunofluorescent labeling of NESTIN, β-TUBULIN III, and VIMENTIN.

Pluripotent stem cells proliferation is associated with placentation in dogs.

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs.

Generation of induced pluripotent stem cells from large domestic animals.

Background: Induced pluripotent stem cells (iPSCs) have enormous potential in developmental biology studies and in cellular therapies. Although extensively studied and characterized in human and murine models, iPSCs from animals other than mice lack reproducible results.

Methods: Herein, we describe the generation of robust iPSCs from equine and bovine cells through lentiviral transduction of murine or human transcription factors Oct4, Sox2, Klf4, and c-Myc and from human and murine cells using similar protocols, even when different supplementations were used.

Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues.

Introduction: Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species.

Objectives: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes.

Methods: Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced into pluripotency (eiPSCs).

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice.

Background: Xenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important.

The timeline development of female canine germ cells.

During the sex differentiation, the primordial germ cells (PGCs) pass through a differentiation, becoming spermatogonial cells in males and oocytes in females. In this phase, there is difference in gene expression and differentiation potency between males and females. Specific cell markers have been essential in the PGC meiosis beginning and become oocyte cells.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments.

Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.

Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O). However, the role of NO in mammalian spermatozoa physiology seems paradoxical.

Dynamics of male canine germ cell development.

Primordial germ cells (PGCs) are precursors of gametes that can generate new individuals throughout life in both males and females. Additionally, PGCs have been shown to differentiate into embryonic germ cells (EGCs) after in vitro culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (Canis lupus familiaris).

In vitro identification of a stem cell population from canine hair follicle bulge region.

Skin is an extensive and easily accessible organ possessing various cell types that are constantly renewed. Previous studies have suggested the presence of a stem cell niche at the bulge region of the hair follicle, which contains cells positive for CD200 and CD34. Thus, this study sought to identify these cell populations in canine skin cells using the following methods 1- collecting samples of adult and fetal skin and isolating and culturing these cells using a method of simple enzymatic digestion and 2- testing the cell cultures for CD200 and CD34 in vitro and comparing them with skin tissue samples (in situ).

Regulation of neural stem cells by choroid plexus cells population.

The choroid plexus is a tissue on the central nervous system responsible for producing cerebrospinal fluid, maintaining homeostasis and neural stem cells support; though, all of its functions still unclear. This study aimed to demonstrate the niches of choroid plexus cells for a better understanding of the cell types and functions, using the porcine as the animal model. The collected material was analyzed by histology, immunohistochemistry, and cell culture.