Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.Materials, Methods & Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy.

The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black-rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10(7) cells (ACP). Thirteen 25-cm(2) culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10(6) cells per milliliter, and placed in liquid nitrogen at -196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture.

Preimplantation genetic diagnosis was carried out for embryonic analysis in a patient with multiple endocrine neoplasia type 1 (MEN1). This is a rare autosomal-dominant cancer syndrome and the patients with MEN1 are characterized by the occurrence of tumors in multiple endocrine tissues, associated with germline and somatic inactivating mutations in the MEN1 gene. This case report documents a successful preimplantation genetic diagnosis (PGD) involving a couple at-risk for MEN1 syndrome, with a birth of a healthy infant. The couple underwent a cycle of controlled ovarian stimulation and intracytoplasmic sperm injection (ICSI). Embryos were biopsied at the blastocyst stage and cryopreserved; we used PCR-based DNA analysis for PGD testing. Only one of the five embryos analyzed for MEN1 syndrome was unaffected. This embryo was thawed and transferred following endometrial preparation. After positive βHCG test; clinical pregnancy was confirmed by ultrasound, and a healthy infant was born. PGD for single gene disorders has been an emerging therapeutic tool for couples who are at risk of passing a genetic disease on to their offspring.

Mesenchymal stem cells have been widely used in the treatment of various chronic diseases. The objective of this survey was to evaluate the therapeutic and regenerative potential of stem cells from adipose tissue (ASCs) in the milk production recovery repair of tissue injury in mastitis goats treated with antimicrobial agents prior to cell therapy. After the diagnosis of mastitis and treatment with gentamicin, eight lactating goats were selected for cellular and subsequent therapy, physical-chemical analysis of milk, ultrasonographic and histopathological examinations. The ASCs were taken from the subcutaneous fat of a young goat cultivated in vitro, marked with Qdots-655 and injected in the left mammary gland, being the right mammary gland used as the control. After 30 days the ultrasonographic and histopathological analyzes were repeated and, in the first lactation period, the physical-chemical analysis of the milk was reapeated. Before the cellular therapy, the physical-chemical quality of the milk was compromised and the ultrasonographic and histopathological analysis revealed a chronic inflammatory process and fibrous tissue. The marking of the ASCs with Qdots enabled the tracking, by fluorescence microscopy (BX41-OLYMPUS), in the mammary tissue. In the ASCs therapy, cultures showed high cellularity and characteristics favorable to preclinical studies; with the therapy the physical-chemical parameters of the milk, fat, protein, temperature and pH showed significant differences among the groups; five animals treated with ASCs reconstituted the functionality of the gland and the connective tissue reduced in quantity and inflammatory infiltrate cells. ASCs have potential for the possible regeneration of fibrous mastitis lesions in the mammary gland, however, it would be necessary to increase injection time for the histopathological analysis, since the reconstitution of the glandular acini within the assessed period was not finalized. ASCs can be used to reestablish milk production in goat with chronic mastitis repair mammary lesions, with potential to be a promising clinical alternative for animal rehabilitation for productivity.

There is a growing interest in the study of unspecialized mesenchymal stem cells, for there are still some discussions about their in vitro behavior. Regenerative medicine is a science undergoing improvement which develops treatments as cell therapy using somatic stem cells. In several studies, adipose tissue is presented as a source of multipotent adult cells that has several advantages over other tissue sources. This study aimed to characterize and evaluate the tagging of mesenchymal stem cells from the agoutis adipose tissue (Dasyprocta prymonolopha), with fluorescent intracytoplasmic nanocrystals. Fibroblast cells were observed, plastic adherent, with extended self-renewal, ability to form colonies, multipotency by differentiation into three lineages, population CD90 + and CD45 - expression, which issued high red fluorescence after the tagging with fluorescent nanocrystals by different paths and cryopreserved for future use. It is possible to conclude that mesenchymal stem cells from agouti adipose tissue have biological characteristics and in vitro behavior that demonstrate its potential for use in clinical tests.

PURPOSE:To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS:Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) -Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers -Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS:Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 10 6 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS:Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied.Key words: Cell Culture Techniques. Cell Differentiation. Stem Cells. Flow Cytometry. Goats. Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil

Objective: Patients submitted to oncological fertility preservation with letrozole and gonadotropins seem to present a higher rate of immature oocytes and lower fertilization rates in comparison to infertile patients submitted to IVF cycles with gonadotropins. The aim of this study was to evaluate the influence of letrozole on oocyte morphology in patients with breast cancer submitted to fertility preservation. Methods: Retrospective analysis performed at a public tertiary hospital in São Paulo, Brazil. The oocytes were retrieved from patients with breast cancer undergoing fertility preservation (n=69), and from infertile women undergoing in vitro fertilization (n=92). We evaluated 750 oocytes obtained from breast cancer patients submitted to ovarian stimulation with letrozole and gonadotropins, and 699 oocytes from patients without breast cancer submitted to ovarian stimulation for in vitro fertilization with gonadotropins only due to male factor infertility. The mature oocytes retrieved were analyzed for the presence of refractile bodies, ooplasm color and regularity, central granulation degree, cortical granules, zona pellucida staining and regularity, perivitelline space, presence of vacuoles or abnormal smooth-surfaced endoplasmic reticle and oocyte retraction. Results: There was a higher incidence of alterations in oocyte morphology in the letrozole group when compared to the control group: increased perivitelline space ( p =0.007), irregular zona pellucida ( p <0.001), refractile bodies ( p <0.001), dark ooplasm ( p <0.001), granular ooplasm ( p <0.001), irregular ooplasm ( p <0.001) and dense central granulation ( p <0.001). Conclusion: Letrozole is a risk factor for worse oocyte morphology. However, the clinical impact of ovarian stimulation protocol with combined use of gonadotropins and letrozole for fertility preservation remains unclear in this setting. These data underline the importance of establishing the predictive potential of morphological dimorphisms of human oocytes in IVF outcomes.