In Vitro Activation and Development of Goat Preantral Follicles Enclosed in Ovarian Tissue Co-cultured with Mesenchymal Stem Cells.

The development of culture systems capable of maintaining follicular growth since the preantral stage has been the target of investigations. Mesenchymal stem cells (MSC) present potential for use in a wide range of applications, including research aimed at preserving fertility. Therefore, this study investigated the use of caprine Wharton's Jelly Mesenchymal Stem Cells (WJMSC) on the survival and in vitro development of goat preantral follicles enclosed in ovarian fragments cultured for 1 or 7 days. Fragments of the ovarian cortex were immediately fixed (non-cultured control) or distributed in four treatments: ovarian tissue cultured in control medium (α-MEM+); ovarian tissue cultured in α-MEM+ supplemented with FBS (α-MEM+ + FBS); ovarian tissue co-cultured with stem cells in α-MEM+ (α-MEM+ + SC); and ovarian tissue co-cultured with stem cell in α-MEM+ + FBS (α-MEM+ + SC + FBS). The rates of cell proliferation, follicular survival, and activation, as well as follicular diameter, were evaluated. After 7 days, the treatment co-cultured with stem cells showed a higher (P < 0.05) percentage of morphologically normal preantral follicles compared to the other treatments, as well as a higher (P < 0.05) activation rate compared to cultured control. Moreover, the follicular diameter was higher (P < 0.05) in the treatment co-cultured with stem cells compared to co-cultured with stem cells plus FBS. This study demonstrates for the first time that in vitro co-culture of caprine WJMSC with preantral follicles enclosed in goat ovarian tissue improves activation and early follicular development.

Adipose stem cells in reparative goat mastitis mammary gland.

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.

Labeling of adipose-derived stem cells with quantum dots provides stable and long-term fluorescent signal for ex vivo cell tracking.

Stem cells derived from adipose tissue (ADSC) have been used in cell therapy as an alternative to treat chronic and degenerative diseases. Using biomedical and image trials to track the cells when infused in the target tissue is essential to control cell migration and adhesion. The objective of the present study was to label and assess the adhesion of goat adipose tissue-derived stem cells (g-ADSC) after cell infusion in animal models by tracking luminescent intracytoplasmatic nanocrystals. The cells were labeled by using Qdots. The g-ADSCs infused with nanocrystal were prepared either fresh or fixed and further visualized under a fluorescence microscope. The labeled cells were infused in the goat mammary glands and mouse testicles and kidneys via tail vein injection. Thirty days after cell infusion, biopsy was carried out for analyses. The g-ADSC cultures were presented with high cellularity and fibroblast morphology, even after infusion of the nanocrystals. It was possible, by processing in paraffin and under fluorescence microscopy, demonstrating the success of the labeling in the long term. Freezing mammary gland biopsies in liquid NO2 did not alter the quality of labeling with Qdots. Therefore, g-ADSCs can be labeled with intracytoplasmatic nanocrystals (Qdots) enabling their in vitro and ex vivo tracking.

Role of the autologous mesenchymal stem cells compared with platelet rich plasma on cicatrization of cutaneous wounds in diabetic mice / Papel das células-tronco mesenquimais autólogas em comparação com plasma rico em plaquetas na cicatrização de feridas cutâneas em camundongos diabéticos

Chronic cutaneous lesions affect 15% of diabetic human patients and represent a risk 15 to 46 times larger of limb amputations compared to people with normal glycemia. It is assumed that half of these amputations could be prevented by early treatment of wounds, for example, with proper cell therapy. Objectives: In this study, the action of the autologous transplant of mesenchymal stem-cells (MSC) was evaluated compared to the treatment with autologous platelet rich plasma (PRP) in the cicatrization of cutaneous lesions induced in diabetic mice. These animals were previously treated with streptozootocin to induce diabetes mellitus and round wounds of 1.5cm in diameter were created in the posterior region. Diameters of the wounds and healing time were evaluated during 30 days and the results were submitted to variance analysis and Tukey's test average. It was noticed that the animals treated with MSC presented a more accelerated cicatrization of the cutaneous lesion than the animals treated with PRP. However, the treatment with PRP presented better results than just the daily asepsis of the lesions with saline or covering them with semi-permeable bandage. Besides, the use of semi-permeable bandage kept the cutaneous lesions of diabetic mice did not interfere negatively with cicatrization, proved to be harmless to use, but kept the cutaneous lesions more hydrated than the ones exposed to the environment.(AU)

Lesões cutâneas crônicas afetam 15% dos pacientes diabéticos e humanos representam um risco 15 a 46 vezes maior de amputações de membros em comparação com as pessoas com a glicemia normal. Supõe-se que a metade destas amputações poderia ser evitada por meio do tratamento precoce das feridas cutâneas com, por exemplo, uma adequada terapia celular. Objetivos: Neste estudo, a ação do transplante autólogo de células estaminais mesenquimais (MSC) foi avaliada em comparação com o tratamento com plasma rico em plaquetas autólogo (PRP) na cicatrização de lesões cutâneas induzidas em camundongos diabéticos. Estes animais foram previamente tratados com estreptozotocina para induzir diabetes mellitus e feridas redondas de 1,5 cm de diâmetro foram criadas na região posterior. Os diâmetros dos ferimentos e tempo de cicatrização foram avaliados durante 30 dias e os resultados foram submetidos à análise de variância e média pelo teste de Tukey. Verificou-se que os animais tratados com MSC apresentam uma cicatrização mais acelerada da lesão cutânea que do que os animais tratados com PRP. No entanto, o tratamento com PRP apresentou melhores resultados do que apenas a assepsia das lesões diariamente com solução salina ou cobrindo-os com atadura semi-permeável. Além disso, a utilização de atadura semi-permeável mantidas as lesões cutâneas de camundongos diabéticos não interfere negativamente com a cicatrização, provou ser inofensiva para usar, mas manteve as lesões cutâneas hidratadas mais do que os expostos ao meio ambiente.(AU)

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831) / Isolamento, expansão e diferenciação de progenitors celulares obtidos da polpa dentária de cutia (Dasyprocta prymnolopha Wagler, 1831)

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.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831). Este estudo teve como objetivo isolar, expandir, diferenciar e caracterizar células progenitoras existentes na polpa dentária de cutia. O material foi lavado em solução de PBS e dissociado mecanicamente, com o auxílio de uma lâmina de bisturi, em placas contendo meio de cultura D-MEM/F-12, e incubadas em 5% de CO2-37⁰C. A curva de crescimento, o ensaio de CFU, a diferenciação osteogênica/adipogênica e a caracterização foram obtidas a partir do isolamento. As células começaram a ser liberadas, a partir do explante, em torno do sétimo dia de cultura. A partir do 22o dia, as células atingiram 80% de confluência. No teste para UFC, 81 colônias foram contadas aos 12 dias de cultivo. As curvas de crescimento pré- e pós-congelamento apresentaram crescimento regular, com intensa proliferação e potencial clonogênico. A diferenciação das células mostrou a formação de osteoblastos e de células de gordura, a partir de 15 dias de cultura em meio específico. A citometria de fluxo (FACS) apresentou-se como segue: CD34 (positivo), CD14 (negativo), CD45 (negativo), CD73 (positivo), CD79 (negativo), CD90 (positivo), CD105 (positivo), demonstrando a grande especificidade e comprometimento das células isoladas com linhagens de células-tronco mesenquimais. Estes resultados sugerem a existência de uma população de células-tronco mesenquimais isolada a partir de explantes da polpa dentária cutia, um modelo potencial para o estudo de linhagens de células-tronco obtidas a partir do tecido pulpar.

Treatment of critical defects produced in calvaria of mice with mesenchymal stem cells.

Mesenchymal stem cells (MSC) are present in specialized niches in perivascular regions of adult tissues and are able to differentiate into various cell types, such as those committed to repairing. Bone marrow derived MSC from eight young mice C57BL/ 6 gfp(+) were expanded in culture for repairing critical defects in calvarial bone produced in twenty-four young isogenic adult C57BL/6 mice. The animals were subjected to a cranial defect of 6.0mm diameter and divided into two equal experimental groups. Control group did not receive any treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL into the defects. The group treated with MSC showed increased angiogenesis and amount of new bone deposited on the defect limits than that observed in the control group. The results demonstrated that transplantation of bone marrow-derived MSC of C57BL/6 gfp(+) mice to bone critical defects produced in mice calvarial contributes positively to the bone repair process. MSC presets ability to influence the correct functioning of osteoblasts, increases the amount of mobilized cells for the repairing process, speeds up growth, and increases deposition of bone matrix.