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. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine.

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.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.

Is dynapenic abdominal obesity a risk factor for cardiovascular mortality? A competing risk analysis.

BACKGROUND: Dynapenic abdominal obesity has been shown as a risk factor for all-cause mortality in older adults. However, there is no evidence on the association between this condition and cardiovascular mortality. OBJECTIVE: We aimed to investigate whether dynapenic abdominal obesity is associated with cardiovascular mortality in individuals aged 50 and older. METHODS: A longitudinal study with an 8-year follow-up was conducted involving 7,030 participants of the English Longitudinal Study of Ageing study. Abdominal obesity and dynapenia were respectively defined based on waist circumference (> 102 cm for men and > 88 cm for women) and grip strength (< 26 kg for men and < 16 kg for women). The sample was divided into four groups: non-dynapenic/non-abdominal obesity (ND/NAO), non-dynapenic/abdominal obesity (ND/AO), dynapenic/non-abdominal obesity (D/NAO) and dynapenic/abdominal obesity (D/AO). The outcome was cardiovascular mortality. The Fine-Grey regression model was used to estimate the risk of cardiovascular mortality as a function of abdominal obesity and dynapenia status in the presence of competing events controlled by socio-demographic, behavioural and clinical variables. RESULTS: The risk of cardiovascular mortality was significantly higher in individuals with D/AO compared with ND/NAO (SHR 1.85; 95% CI: 1.15-2.97). D/NAO was also associated with cardiovascular mortality (SHR: 1.62; 95% CI: 1.08-2.44). CONCLUSION: Dynapenic abdominal obesity is associated with cardiovascular mortality, with a larger effect size compared to dynapenia alone in individuals older than 50 years. Thus, prevention strategies and clinical interventions that enable mitigating the harmful effects of these conditions should be adopted to diminish such risk.

Does the incidence of frailty differ between men and women over time?

BACKGROUND/OBJECTIVE: The mechanisms, risk factors and influence of sex on the incidence of frailty components are not fully understood. The aim of this study was to analyse sex differences in factors associated with the increase in the number of frailty components. METHODS: A 12-year follow-up analysis was conducted with 1,747 participants aged ≥ 60 of the ELSA Study with no frailty at baseline. Generalised linear mixed models were used to analyse the increase in the number of frailty components stratified by sex, considering socioeconomic, behavioural, clinical and biochemical characteristics as exposure variables. RESULTS: The increase in the number of frailty components in both sexes was associated with an advanced age (70 to 79 years and 80 years or older), low educational level, sedentary lifestyle, elevated depressive symptoms, joint disease, high C-reactive protein levels, perception of poor vision and uncontrolled diabetes (p < 0.05). Osteoporosis, low weight, heart disease, living with one or more people and perception of poor hearing were associated with an increase in the number of frailty components in men. High fibrinogen concentration, controlled diabetes, stroke and perception of fair vision were associated with the outcome in women (p < 0.05). Obese women and men and overweight women had a lower increase in the number of frailty components compared to those in the ideal weight range. CONCLUSIONS: Socioeconomic factors, musculoskeletal disorders, heart disease and low weight seem to sustain the frailty process in men, whereas cardiovascular and neuroendocrine disorders seem to sustain the frailty process in women.

Gender differences in the association between adverse events in childhood or adolescence and the risk of premature mortality.

To examine, by gender, the relationship between adverse events in childhood or adolescence and the increased risk of early mortality (before 80 years). The study sample included 941 participants of the English Longitudinal Study of Aging who died between 2007 and 2018. Data on socioeconomic status, infectious diseases, and parental stress in childhood or adolescence were collected at baseline (2006). Logistic regression models were adjusted by socioeconomic, behavioral and clinical variables. Having lived with only one parent (OR 3.79; p = 0.01), overprotection from the father (OR 1.12; p = 0.04) and having had an infectious disease in childhood or adolescence (OR 2.05; p = 0.01) were risk factors for mortality before the age of 80 in men. In women, overprotection from the father (OR 1.22; p < 0.01) was the only risk factor for mortality before the age of 80, whereas a low occupation of the head of the family (OR 0.58; p = 0.04) and greater care from the mother in childhood or adolescence (OR 0.86; p = 0.03) were protective factors. Independently of one's current characteristics, having worse socioeconomic status and health in childhood or adolescence increased the risk of early mortality in men. Parental overprotection increased the risk of early mortality in both sexes, whereas maternal care favored longevity in women.

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. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

Are Serum 25-Hydroxyvitamin D Deficiency and Insufficiency Risk Factors for the Incidence of Dynapenia?

Epidemiological evidence showing the association between low 25(OH)D and age-related reduction in neuromuscular strength (dynapenia) is a paucity and controversial and, to date, the effect of osteoporosis and vitamin D supplementation on these associations has not been measured. Thus, we analyze whether serum 25(OH)D deficiency and insufficiency are risk factors for the incidence of dynapenia in individuals aged 50 or older and whether osteoporosis or vitamin D supplementation modify these associations. For that, 3205 participants of the ELSA study who were non-dynapenic at baseline were followed for 4 years. Vitamin D was measured at baseline by the serum concentration of 25(OH)D and classified as sufficient (> 50 nmol/L), insufficient (≥ 30 and ≤ 50 nmol/L) or deficient (< 30 nmol/L). The incidence of dynapenia was determined by a grip strength < 26 kg for men and < 16 kg for women at the end of the 4-year follow-up. Poisson regression models were adjusted by sociodemographic, behavioral, clinical and biochemical characteristics. Serum 25(OH)D deficient was a risk factor for the incidence of dynapenia (IRR = 1.70; 95% CI 1.04-2.79). When only individuals without osteoporosis and those who did not use vitamin D supplementation were analyzed, both serum 25(OH)D deficiency (IRR = 1.78; 95% CI 1.01-3.13) and insufficiency (IRR = 1.77; 95% CI 1.06-2.94) were risk factors for the incidence of dynapenia. In conclusion, a serum level of 25(OH)D < 30 nmol/L is a risk factor for the incidence of dynapenia. Among individuals without osteoporosis and those who do not take vitamin D supplementation, the threshold of risk is higher (≤ 50 nmol/L).

Sex Differences in Vitamin D Status as a Risk Factor for Incidence of Disability in Instrumental Activities of Daily Living: Evidence from the ELSA Cohort Study.

Vitamin D deficiency compromises elements underlying the disability process; however, there is no evidence demonstrating the association between vitamin D deficiency and the incidence of disability in instrumental activities of daily living (IADL). We investigated the association between vitamin D deficiency and the risk of incidence of IADL disability separately in men and women. A total of 4768 individuals aged ≥50 years from the English Longitudinal Study of Aging (ELSA) and without IADL disability according to the Lawton scale were available. Vitamin D was evaluated at baseline by serum 25(OH)D concentrations and classified as sufficient (>50 nmol/L), insufficient (>30 to ≤50 nmol/L) or deficient serum (≤30 nmol/L). IADL were reassessed after 4 years. Poisson models stratified by sex and controlled by covariates demonstrated that deficient serum 25(OH)D was a risk factor for the incidence of IADL disability in men (IRR: 1.43; 95% CI 1.02, 2.00), but not in women (IRR: 1.23; 95% CI 0.94, 1.62). Men appear to be more susceptible to the effect of vitamin D deficiency on the incidence of IADL disability, demonstrating the importance of early clinical investigation of serum 25(OH)D concentrations to prevent the onset of disability.

In vitro 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 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.

Combination of dynapenia and abdominal obesity affects long-term physical performance trajectories in older adults: sex differences.

BACKGROUND: There is little epidemiological evidence of sex differences in the association between dynapenic abdominal obesity and the decline in physical performance in older adults. OBJECTIVES: The aims of the present study were to investigate whether the decline in physical performance is worse in individuals with dynapenic abdominal obesity and whether there are sex differences in this association. METHODS: Of 6183 individuals aged ≥60 y from the English Longitudinal Study of Ageing, 2308 participants with missing data were excluded. Therefore, a longitudinal analysis was conducted with 3875 older adults. Abdominal obesity was determined based on waist circumference (>102 cm for males, and >88 cm for females), and dynapenia was based on grip strength (<26 kg for males, <16 kg for female). The sample was divided into 4 groups: nondynapenic/nonabdominal obesity (ND/NAO), nondynapenic/abdominal obesity (ND/AO), dynapenic/nonabdominal obesity (D/NAO), and dynapenic/abdominal obesity (D/AO). Decline in physical performance in an 8-y follow-up period was analyzed using generalized linear mixed models. RESULTS: At baseline, both male (-1.11 points; 95% CI: -1.58, -0.65 points; P < 0.001) and female (-1.39 points; 95% CI: -1.76, -1.02 points; P < 0.001) with D/AO had worse performances on the Short Physical Performance Battery (SPPB) than their counterparts in the ND/NAO group. Over the 8-y follow-up, males with D/AO had a faster rate of decline in the SPPB performance compared with males in the ND/NAO group (-0.11 points/y; 95% CI: -0.21, -0.01 points; P = 0.03). CONCLUSIONS: D/AO is associated with a stronger decline in physical performance in males but not in females. The identification and management of dynapenic abdominal obesity could be essential to avoiding the first signs of functional impairment in older males.

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. Continuous bFGF supplementation during piPSCs culture was beneficial to support a pluripotent state and the differentiation of piPSCs into pPGCLCs. The pPGCLCs were positive for the gene and protein expression of pluripotent and germinative markers. This study can provide a suitable in vitro model for use in translational studies and to help answer numerous remaining questions about germ cells.

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. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

Is slowness a better discriminator of disability than frailty in older adults?

BACKGROUND: The trajectory of incident disability that occurs simultaneously with changes in frailty status, as well as how much each frailty component contributes to this process in the different sexes, are unknown. The objective of this study is to analyse the trajectory of the incidence of disability on basic and instrumental activities of daily living (BADL and IADL) as a function of the frailty changes and their components by sex over time. METHODS: Longitudinal analyses of 1522 and 1548 of the English Longitudinal Study of Ageing study participants without BADL and IADL disability, respectively, and without frailty at baseline. BADL and IADL were assessed using the Katz and Lawton Scales and frailty by phenotype at 4, 8, and 12 years of follow-up. Generalized mixed linear models were calculated for the incidence of BADL and IADL disability, as an outcome, using changes in the state of frailty and its components, as the exposure, by sex in models fully adjusted for sociodemographic, behavioural, biochemical, and clinical characteristics. RESULTS: The mean age, at baseline, of the 1522 eligible individuals free of BADL and free of frailty was 68.1 ± 6.2 years (52.1% women) and of the 1548 individuals free IADL and free frailty was 68.1 ± 6.1 years (50.6% women). Women who became pre-frail had a higher risk of incidence of disability for BADL and IADL when compared with those who remained non-frail (P < 0.05). Men and women who became frail had a higher risk of incidence of disability regarding BADL and IADL when compared with those who remained non-frail (P < 0.05). Slowness was the only component capable of discriminating the incidence of disability regarding BADL and IADL when compared with those who remained without slowness (P < 0.05). Weakness and low physical activity level in men and exhaustion in women also discriminated the incidence of disability (P < 0.05). CONCLUSIONS: Slowness is the main warning sign of functional decline in older adults. As its evaluation is easy, fast, and accessible, screening for this frailty component should be prioritized in different clinical contexts so that rehabilitation strategies can be developed to avoid the onset of disability.

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 ß. The FSH ß-subunit (FSHß) 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 ß-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.

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). 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.

Dynapenia, abdominal obesity or both: which accelerates the gait speed decline most?

OBJECTIVE: to investigate whether the combination of dynapenia and abdominal obesity is worse than these two conditions separately regarding gait speed decline over time. METHODS: a longitudinal study was conducted involving 2,294 individuals aged 60 years or older free of mobility limitation at baseline (gait speed >0.8 m/s) who participated in the English Longitudinal Study of Ageing. Dynapenia was determined as a grip strength <26 kg for men and <16 kg for women. Abdominal obesity was determined as a waist circumference >102 cm for men and >88 cm for women. The participants were divided into four groups: non-dynapenic/non-abdominal obese (ND/NAO); only abdominal obese (AO); only dynapenic (D) and dynapenic/abdominal obese (D/AO). Generalised linear mixed models were used to analyse gait speed decline (m/s) as a function of dynapenia and abdominal obesity status over an 8-year follow-up period. RESULTS: over time, only the D/AO individuals had a greater gait speed decline (-0.013 m/s per year, 95% CI: -0.024 to -0.002; P < 0.05) compared to ND/NAO individuals. Neither dynapenia nor abdominal obesity only was associated with gait speed decline. CONCLUSION: dynapenic abdominal obesity is associated with accelerated gait speed decline and is, therefore, an important modifiable condition that should be addressed in clinical practice through aerobic and strength training for the prevention of physical disability in older adults.

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. 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.

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, ß-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.

Neurons-derived extracellular vesicles promote neural differentiation of ADSCs: a model to prevent peripheral nerve degeneration.

Potential mechanisms involved in neural differentiation of adipocyte derived stem cells (ADSCs) are still unclear. In the present study, extracellular vesicles (EVs) were tested as a potential mechanism involved in the neuronal differentiation of stem cells. In order to address this, ADSCs and neurons (BRC) were established in primary culture and co-culture at three timepoints. Furthermore, we evaluated protein and transcript levels of differentiated ADSCs from the same timepoints, to confirm phenotype change to neuronal linage. Importantly, neuron-derived EVs cargo and EVs originated from co-culture were analyzed and tested in terms of function, such as gene expression and microRNA levels related to the adult neurogenesis process. Ideal neuron-like cells were identified and, therefore, we speculated the in vivo function of these cells in acute sciatic nerve injury. Overall, our data demonstrated that ADSCs in indirect contact with neurons differentiated into neuron-like cells. Neuron-derived EVs appear to play an important role in this process carrying SNAP25, miR-132 and miR-9. Additionally, in vivo neuron-like cells helped in microenvironment modulation probably preventing peripheral nerve injury degeneration. Consequently, our findings provide new insight of future methods of ADSC induction into neuronal linage to be applied in peripheral nerve (PN) injury.

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. 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. METHODS: SSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L-1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis. RESULTS: Supplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice. CONCLUSIONS: In conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.