differentiation-porcine-induced-pluripotent-stem-cells-pipscs-into

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

São Paulo State University

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo.

New approaches regarding the in vitro maturation of oocytes: manipulating cyclic nucleotides and their partners in crime.

In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus-oocyte complexes and embryos.

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.

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.

Ramon Cesar Botigelli

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)

Ramon Cesar Botigelli

.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