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 in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

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). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

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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). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i—bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

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.

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.

Different supplements are used during invitro cellular reprogramming, usually acting on pluripotency maintenance and/or differentiation inhibition, such as basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and 2i (MEK inhibitor: PD0325901 + GSK3 inhibitor: CHIR99021). Another important factor affecting the reprogramming process is the oxygen (O2) tension because O2 levels can modify cellular metabolism and epigenetic markers, which are known to modulate pluripotency. Our objective was to evaluate the efficiency of reprogramming bovine fibroblasts in combination with different oxygen tensions (high O2, hO2×low O2, lO2) in different cell differentiation inhibitors: bFGF and bFGF + LIF + 2i (FL2i). Bovine fibroblasts were transduced with lentivirus harbouring mouse OSKM transcription factors (OCT4, SOX2, KLF4, and cMYC). Three clonal lineages were analysed for each experimental group. Pluripotency was characterised by morphology, detection of alkaline phosphatase, formation of embryoid bodies, and analysis of gene expression. As an initial pluripotency test, all colonies were positive for alkaline phosphatase (AP) activity in passages 5 to 6. Colonies were cultured for at least 15 passages (±140 days) with the exception of bFL2i colonies cultured in lO2, which did not grow beyond 7 to 8 passages. For gene expression analysis, samples of each colony in passages 5, 10, and 15 were used. When gene expression was analysed, both endogenous NANOG and OCT4 were increased in the bFGF group when cultured in hO2, and bFGF cultured in lO2 was higher than in the FL2i group (P<0.05). Also, NANOG was increased in early passages compared with late passages (P<0.05); SOX2 and FGF5 were increased in lO2 groups (P<0.05). The bFGF treatment increased STELLA expression compared with bFL2i (P<0.05) at both oxygen tensions. Interestingly, exogenous vector expression increased in the bFGF group compared with bFL2i (P<0.05) but was not affected by oxygen tension (P>0.05). All colonies tested were able to form embryoid bodies. In conclusion, it was not possible to maintain bovine induced pluripotent stem cells (biPS) in bFL2i treatment cultured in lO2 because these colonies were not able to remain viable after 8 passages. Moreover, small molecule supplementation strongly affected pluripotency gene expression. Further analysis on epigenetic changes, metabolism, and self-renewal is necessary to understand the pluripotent state in biPS under our experimental conditions. We acknowledge FAPESP for funding (grant 2015/26816-5 and fellowship 2018/24520-7).

The reprogramming process suffers from effects of external and internal factors, one of which is oxygen concentration, because it plays important roles in several processes of the cell. The objective of this study was to evaluate the effect of oxygen during reprogramming of equine adult fibroblasts. Fibroblasts were isolated from the skin of one male and immediately cultured under high (20%, HO group) or low (5%, LO group) oxygen tension. Generation of induced pluripotent stem cells (iPSCs) was performed by transduction with human sequences of OCT4, SOX2, KLF4, and c-MYC cDNAs. Morphology evaluation and RNA samples collection were performed at the day of transduction (Day 0), and then every 3 days (Days 3, 6, 9, 12, 15, and 18). Six days after transduction, cells were passed to mouse embryonic fibroblast (MEFs). A third group, in which the cells were cultured in high oxygen and passed to low oxygen, was formed (high to low group, HLO) and analysed. The equine (e)iPS colonies were evaluated in terms of reprogramming efficiency, morphology, detection of alkaline phosphatase, and qRT-PCR for markers of pluripotency (OCT4, REX1, NANOG, and SOX2 genes), glycolysis (PFKM and GAPDH), mitochondrial fission and fusion (DNM1L and MFN1), hypoxia (HIF1α, HIF2α, and VEGFA), and presence of the exogenous vector (HS). The values of the target genes were normalized by the average values of the housekeeping genes (HPRT1 and PPIA) and the fold changes were calculated using the 2(−ΔCT) equation. After 10, 11 and 14 days of transduction, the first colonies appeared in the LO, HO, and HLO groups. Their reprogramming efficiency was 1.67%, 0.08%, and 0.025%, respectively. The cells were primarily identified by their morphology: colonies with well-defined edges and cells with a high nuclear/cytoplasm ratio. Our results indicated that genes related to pluripotency increased at first, and then some decreased at Day 18, probably due to the time of picking the first colonies. The LO group showed more elevated levels of these genes than HO group, in accordance with the higher reprogramming efficiency found in LO. Lentiviral vector expression peaked at Day 3 and then decreased at Day 3 in LO and at Day 12 in HO. Glycolysis genes were less expressed in LO, which may suggest a successful metabolism change. The mitochondrial gene MF1 was higher in HO than in LO, and expression of DNM1L increased at Day 12 in the LO group. Expression of VEGFA, a direct target of HIF1α, behaved similarly. Expression of HIF1α and HIF2α differed from expected, being higher in LO than in HO. The expression of HIF1α and HIF2α increased when the shift from high to low oxygen occurred, suggesting that these genes were not expressed before, likely because cells were adapted to the low oxygen tension. When the shift occurs, the cells reacted with an increase of HIF1α and HIF2α. The study of these effects during reprogramming invitro is critical, and biological repetitions to reinforce this data are underway. Herein, we show that the invitro reprogramming process may be modulated by environmental oxygen changes.