Summary 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.
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). Immunofluorescence results were negative for both CD200 and CD34 in frozen and paraffin embedded tissue, whereas the analysis showed that cultured cells positive for CD34, CD200 and double positive cells could be visualized in different percentages. Additionally, the pluripotency marker OCT4 was positive in the isolated cells. Analysis of CD34, CD200 and OCT4 by RT-qPCR showed that there is expression in fetal and adult cells, although no difference was observed between groups. Our results suggest that bulge stem cells from both fetuses and adult dogs were reported with the use of CD34 and CD200 markers in this study, and further techniques for cell isolation and in vitro cultivation are needed in order to obtain enriched populations of skin stem cells in dogs.
The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.
This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.
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.
The aim of this study was to evaluate the effect of epidural bupivacaine administration at the first lumbar vertebra on cardiopulmonary variables, arterial blood gases and anti-nociception. Sixteen healthy female dogs were randomly assigned into two groups based on bupivacaine dose: G1 group, 1mg kg -1 or G2 group, 2mg kg ). Cardiopulmonary variables were measured and arterial blood gas was collected (T0), it was repeated 10 minutes after intravenous administration of butorphanol 0.4mg kg -1 (T1). Anesthesia was induced with intravenous etomidate at 2mg kg -1 and the epidural catheter was introduced and placed at the first lumbar vertebra. Thirty minutes later, bupivacaine was administered epidurally. Cardiopulmonary measurements and arterial blood gas analysis were recorded at 10 minute intervals (T2 to T6). Evaluation of pre surgical anti-nociception was performed at 5 minute intervals for 30 minutes by clamping the hind limbs, anus, vulva, and tail with the dogs awake. Subsequently, ovariohysterectomy was performed and adequacy of surgical anti-nociception was evaluated at 5 time points. Parametric data were analyzed using the F test with a <0.05 significance. After bupivacaine administration, there were differences between groups just for bicarbonate means (HCO 3 -) on T6 (P=0.0198), with 18.7±1.3 and 20.4±0.8 for G1 and G2, respectively. After T1, before bupivacaine administration, both groups presented a slightly lower pH, base excess (BE), the end-tidal carbon dioxide tension (PECO 2 ), and partial pressure of carbon dioxide (PaCO 2 ), suggesting mild metabolic acidosis. G2 showed better antinociceptive effect both before and during surgery. It was possible to perform ovariohysterectomy in 87.5% of the G2 bitches and 25% of the G1 bitches. The two doses of bupivacaine evaluated do not cause important alterations in the studied parameters and the dose of 2mg kg -1 results in a better antinociceptive effect.Keywords: bupivacaine, dog, epidural catheter, regional anesthesia RESUMO O objetivo deste estudo foi avaliar os efeitos da administração epidural de bupivacaína à altura da primeira vértebra lombar sobre variáveis cardiopulmonares, hemogasometria arterial e antinocicepção. Dezesseis cadelas foram separadas aleatoriamente em dois grupos que se diferenciaram pela dose de bupivacaína, 1mg/kg (G1) ou 2mg/kg (G2), diluídas no mesmo volume final (1mL/4kg). As variáveis cardiopulmonares e hemogasometria arterial foram coletadas antes (T0) e após 10 minutos da administração intravenosa de 0,4mg/kg de butorfanol (T1). A anestesia foi induzida com 2mg/kg de etomidato intravenoso para introdução do cateter epidural. Após 30 minutos, a bupivacaína foi administrada e, passados 10 minutos, nova coleta de parâmetros foi feita, sendo repetida a cada 10 minutos (T2 a T6
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 in vivo and offspring analysis; therefore, suitable for both medicine and animal production. AIM To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine. METHODS The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation. RESULTS The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts. CONCLUSION For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.
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