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 ...

University of São Paulo

This study complements the previous investigations of the reproductive biology of male viscachas, a rodent of a seasonal Hystricognathi that exhibits photoperiod-induced morphological variations in the reproductive system. In the present study, a quantitative analysis of spermatogenesis was performed during the summer and the spring. Spermatogonial cells were analyzed to determine by immunolabelling for STRA8 and DAZL, which are essential for spermatogenesis. Six free-living male viscachas were captured, three animals in the summer during the period of reproductive activity and three animals in the spring during the period of testicular regression. The testes of the viscachas were collected and processed for light microscopy, macroscopic and immunochemical analyses. The germ and Sertoli cells present in the seminiferous tubules were quantitatively analyzed in each animal. The efficiency coefficient for spermatogonial mitosis, meiotic yield, overall spermatogenesis yield and Sertoli cell index, revealed that the Sertoli cells in male viscachas captured during the summer had a reduced capacity to structurally and nutritionally support the developing round spermatids compared with the male viscachas captured during the spring. The animals produced less sperm during the spring than the summer, suggesting a seasonal impact on spermatogenesis. Immunolabelling for STRA8 and DAZL was detected during summer and spring seasons. These results suggest that in seasonal rodents, such as the male viscachas, the photoperiod promotes significant changes in the testis and in the germ cell yield.

Seasonal variations cause morphological changes and altered spermatogenesis in the testes of viscacha (Lagostomus maximus).

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. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.

Stem cells on regenerative and reproductive science in domestic animals.

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. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.

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

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.

Generation of induced pluripotent stem cells from large domestic animals.

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. NPC-like cells were positive for all the neural markers tested. These results evidence of the generation of porcine NPC-like cells after in vitro induction with chemical inhibitors, representing an adequate model for future regenerative and translational medicine research.

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

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.

Naira Caroline Godoy Pieri

Department of Veterinary Medicine,
Faculty of Animal Sciences and Food Engineering,
Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering

University of São Paulo

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

Naira Caroline Godoy Pieri

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Veterinary Medicine,Faculty of Animal Sciences and Food Engineering,Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering

University of São Paulo

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

Department of Veterinary Medicine,
Faculty of Animal Sciences and Food Engineering,
Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering