Mfsd2a Promotes the Proliferation, Migration, Differentiation and Adipogenesis of Chicken Intramuscular Preadipocytes

Intramuscular fat (IMF) content is a crucial parameter for estimating meat quality. Growing evidence indicates that gene regulation plays an important role in IMF deposition. This study aimed to determine the function of Mfsd2a in chicken intramuscular preadipocytes. In the present study, high Mfsd2a mRNA levels were observed in the liver and adipose tissues of broilers. Subsequently, we synthesized small interfering RNAs to silence the expression of Mfsd2a in chicken intramuscular preadipocytes. The following results suggested that CDK2, PCNA, CCND1, CCND2 and MKI67 were inhibited, with CCK-8 and EdU assays revealing that cell proliferation was inhibited. Scratch test showed that cell migration ratios were declined. We also found that Mfsd2a silencing decreased the mRNA levels of PPARγ, RXRG and their target genes. The similar results were found in some key genes that contribute to lipid synthesis, including C/EBPα, C/EBPß, FABP4, FASN, ACACA and ACSL1. Finally, Oil red O staining showed that IMF accumulation was blocked after Mfsd2a silencing. In conclusion, our results implied that Mfsd2a promotes the proliferation and migration of chicken intramuscular preadipocytes, as well as the differentiation and adipogenesis through PPARγ signaling pathway, which may provide a potential target to improve chicken meat quality.(AU)

Prenatal origins of productivity and quality of beef

The productive traits of beef cattle are orchestrated by their genetics, postnatal environmental conditions, and also by the intrauterine background. Both under- or overnutrition, as specific dietary components, are able to promote persistent effects on the offspring. This occurs because dietary factors act not only affecting the availability of substrates for fetal anabolism and oxidative metabolism, but also as signals that regulate several events toward fetal development. Therefore, this study aimed to summarize the gestational nutrition effects on the offspring performance and meat quality in a long term. Overall, studies have shown that many of these alterations are under the control of epigenetic mechanisms, as DNA methylation, histones modification, and non-coding RNA. The current knowledge has indicated that the fetal programming responses are dependent on the window of fetal development in which the dietary treatment is applied, the intensity of maternal nutritional stimuli, and the treatment application length. Collectively, studies demonstrated that muscle cell hyperplasia is impaired when maternal requirements were not achieved in the second third of gestation, which limits the formation of a greater number of muscle fibers and the offspring growth potential in a long term. Changes in muscle fibers metabolism and in collagen content were also reported as consequence of a dietary perturbation during pregnancy. In contrast, a maternal overnutrition during the late pregnancy has been associated with beneficial responses on meat quality. In summary, ensuring an adequate maternal environment during the fetal development is crucial to enhance the productive responses in beef cattle operations.(AU)

Potential for in vitro mesoderm differentiation of Wharton's jelly cells from ovine umbilical cord isolated in different culture media / Potencial de diferenciação in vitro de células provenientes da geleia de Wharton de cordões umbilicais ovinos isolados em diferentes meios de cultivo

The mammalian Wharton's jelly of umbilical cord (WJUC) is a promising source of multipotent cells, providing advantages due to ethical implications, ease of collection and the absence of teratomas in pre-clinical trials. Ovine multipotent cells have already been isolated from various tissues, however there are no reports using umbilical cords in this species. This study aimed to investigate the best medium to transport the umbilical cord, to isolate and maintain ovine WJUC cells and to compare in vitro growth and mesodermal differentiation potential. Eight ovine umbilical cords were obtained during parturition, sectioned and transported in six different media: MEM, low glucose DMEM, M199, RPMI 1640, PBS and saline. For each transportation medium, four culture media were used and the tissue was explanted in 24-well plates and cultured in MEM, low glucose DMEM, M199 and RPMI 1640, all with 10% FBS. Every experiment was conducted with low-passage (P2), investigating MTT viability during four days and adipogenic, chondrogenic and osteogenesis differentiation was induced in vitro. The most effective transport medium (p<0.1) was low glucose DMEM. There was no bacterial or fungal contamination from collection. Cells from Wharton's jelly of ovine umbilical cords collected at natural birth possess fibroblastic morphology and the capacity for in vitro differentiation into adipogenic, chondrogenic and osteogenic cell lines. MTT tests and in vitro differentiation experiments revealed that cell culture medium modulates the behavior of cells and is an important factor for proliferation and maintenance of multipotency. Low glucose DMEM was the most suitable medium for the isolation of cells from Wharton's jelly of ovine umbilical cord.(AU)

A geleia de Wharton do cordão umbilical (GWCU) de mamíferos é uma fonte promissora de células multipotentes, sendo vantajosa por aspectos éticos, facilidade de coleta e não causar teratomas em ensaios pré-clínicos. Em ovinos, células multipotentes já foram isoladas de vários tecidos, no entanto, não existem relatos do isolamento a partir do cordão umbilical nesta espécie. O objetivo do presente estudo foi investigar o melhor meio para o transporte do cordão umbilical, isolar e manter as células da GWCU ovino em diferentes meios e comparar a proliferação e o potencial de diferenciação mesodermal in vitro. Oito cordões umbilicais foram obtidos, por ocasião do parto natural, seccionados e transportados em seis diferentes meios que consistiram em MEM, DMEM baixa glicose, M199, RPMI 1640, PBS e soro fisiológico. Para cada meio de transporte foram utilizados quatro meios de cultivo, sendo o tecido explantado em placas de 24 poços e cultivados em MEM, DMEM baixa glicose, M199 e RPMI 1640, todos com 10% SFB. Todo o experimento foi realizado em baixa passagem (P2) investigando viabilidade pelo MTT por quatro dias além da indução à diferenciação adipogênica, condrogênica e osteogênica in vitro. O meio de transporte mais efetivo (P<0,10) foi o DMEM baixa glicose. Não houve contaminações bacterianas ou fúngicas decorrentes da coleta. Células oriundas da geleia de Wharton do cordão umbilical ovino colhido por ocasião do parto natural possuem morfologia fibroblastóide e capacidade de diferenciação in vitro nas linhagens adipogênica, condrogênica e osteogênica. Os ensaios de MTT e diferenciação in vitro, revelaram que o meio de cultura celular modula o comportamento destas células, sendo um fator importante tanto para a proliferação como para a manutenção da multipotência, destacando o DMEM baixa glicose como o meio mais adequado para o transporte e isolamento de células da geleia de Wharton do cordão umbilical ovino.(AU)

Potential for in vitro mesoderm differentiation of Wharton's jelly cells from ovine umbilical cord isolated in different culture media / Potencial de diferenciação in vitro de células provenientes da geleia de Wharton de cordões umbilicais ovinos isolados em diferentes meios de cultivo

The mammalian Wharton's jelly of umbilical cord (WJUC) is a promising source of multipotent cells, providing advantages due to ethical implications, ease of collection and the absence of teratomas in pre-clinical trials. Ovine multipotent cells have already been isolated from various tissues, however there are no reports using umbilical cords in this species. This study aimed to investigate the best medium to transport the umbilical cord, to isolate and maintain ovine WJUC cells and to compare in vitro growth and mesodermal differentiation potential. Eight ovine umbilical cords were obtained during parturition, sectioned and transported in six different media: MEM, low glucose DMEM, M199, RPMI 1640, PBS and saline. For each transportation medium, four culture media were used and the tissue was explanted in 24-well plates and cultured in MEM, low glucose DMEM, M199 and RPMI 1640, all with 10% FBS. Every experiment was conducted with low-passage (P2), investigating MTT viability during four days and adipogenic, chondrogenic and osteogenesis differentiation was induced in vitro. The most effective transport medium (p<0.1) was low glucose DMEM. There was no bacterial or fungal contamination from collection. Cells from Wharton's jelly of ovine umbilical cords collected at natural birth possess fibroblastic morphology and the capacity for in vitro differentiation into adipogenic, chondrogenic and osteogenic cell lines. MTT tests and in vitro differentiation experiments revealed that cell culture medium modulates the behavior of cells and is an important factor for proliferation and maintenance of multipotency. Low glucose DMEM was the most suitable medium for the isolation of cells from Wharton's jelly of ovine umbilical cord.(AU)

A geleia de Wharton do cordão umbilical (GWCU) de mamíferos é uma fonte promissora de células multipotentes, sendo vantajosa por aspectos éticos, facilidade de coleta e não causar teratomas em ensaios pré-clínicos. Em ovinos, células multipotentes já foram isoladas de vários tecidos, no entanto, não existem relatos do isolamento a partir do cordão umbilical nesta espécie. O objetivo do presente estudo foi investigar o melhor meio para o transporte do cordão umbilical, isolar e manter as células da GWCU ovino em diferentes meios e comparar a proliferação e o potencial de diferenciação mesodermal in vitro. Oito cordões umbilicais foram obtidos, por ocasião do parto natural, seccionados e transportados em seis diferentes meios que consistiram em MEM, DMEM baixa glicose, M199, RPMI 1640, PBS e soro fisiológico. Para cada meio de transporte foram utilizados quatro meios de cultivo, sendo o tecido explantado em placas de 24 poços e cultivados em MEM, DMEM baixa glicose, M199 e RPMI 1640, todos com 10% SFB. Todo o experimento foi realizado em baixa passagem (P2) investigando viabilidade pelo MTT por quatro dias além da indução à diferenciação adipogênica, condrogênica e osteogênica in vitro. O meio de transporte mais efetivo (P<0,10) foi o DMEM baixa glicose. Não houve contaminações bacterianas ou fúngicas decorrentes da coleta. Células oriundas da geleia de Wharton do cordão umbilical ovino colhido por ocasião do parto natural possuem morfologia fibroblastóide e capacidade de diferenciação in vitro nas linhagens adipogênica, condrogênica e osteogênica. Os ensaios de MTT e diferenciação in vitro, revelaram que o meio de cultura celular modula o comportamento destas células, sendo um fator importante tanto para a proliferação como para a manutenção da multipotência, destacando o DMEM baixa glicose como o meio mais adequado para o transporte e isolamento de células da geleia de Wharton do cordão umbilical ovino.(AU)

Isolation, expansion, differentiation and growth kinetics essay in mesenchymal stem cells culture from the bone marrow of collared peccaries (Tayassu tajacu)

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. [...](AU)

Development of organs and fat deposition in indigenous goat kids under feed restriction / Desenvolvimento dos órgãos e deposição de gorduras em cabritos Canindé sob restrição alimentar

The study aimed to evaluate the effect of feed restriction on the development of organs and fat deposition in castrated Caninde goats. Twenty one goats were distributed in a completely randomized design, with initial body weight of 15.9 ± 1.03kg. The goats were allocated into three levels of feed restriction (seven animals per level): ad libitum; moderate restriction (restriction of 20% on the amount of feed consumed by the animals fed ad libitum) and severe restriction (40% restriction on the amount of feed consumed by the animals fed ad libitum). The diet consisted of 55% forage (Tifton) and 45% concentrate. At 110 days of experiment, the goats were slaughtered with abody weight of 23.5kg ± 2.5kg. The components included the internal organs (spleen, heart, liver, pancreas, lungs, kidneys, blood) the emptied and cleaned digestive tract and fat deposits (in heart, mesenteric, omental and pelvic-renal) were weighed. Feed restriction in Canindé goats affected the weight of organs and fat deposition in regions more involved with the energy storage function (mesenteric, omental and pelvic-renal) (P 0.05), however, feed restriction does not affect the percentage of the organs in relation to EBW (P > 0.05), indicating that even under feed restriction organ development is proportional to the development of the body.(AU)

Objetivou-se avaliar o efeito da restrição alimentar sobre o desenvolvimento dos órgãos e deposição de gordura em caprinos Canindé castrados. Foram utilizados 21 cabritos confinados, em um delineamento inteiramente casualizado, com peso inicial de 15,9 ± 1,03kg. Os cabritos foram alocados em três níveis de restrição alimentar (sete animais por nível): ad libitum (alimentados à vontade); restrição moderada (restrição de 20% em relação à quantidade de matéria natural consumida pelos animais alimentados ad libitum) e restrição severa (restrição de 40% em relação à quantidade de ração consumida pelos animais alimentados ad libitum). A ração experimental apresentou uma proporção de 55% de volumoso (Tifton) e 45% de concentrado. Aos 110 dias de experimento os cabritos foram abatidos com peso médio de 23,5 kg ± 2,5 kg. Esvaziou-se o trato gastrointestinal (TGI), a bexiga e a vesícula biliar e foram mensurados os seus pesos para determinação do peso de corpo vazio (PCV). Foram separados e registrados os pesos dos órgãos (baço, coração, fígado, pâncreas, pulmões, rins, sangue, TGI) e dos depósitos de gordura (cardíaca, mesentérica, omental e pélvico-renal). A restrição alimentar em caprinos Canindé afetou o peso absoluto dos órgãos e das gorduras (P 0.05) que estão mais envolvidas com a função de reserva energética (mesentérica, omental e pélvico-renal), no entanto, não afetou o pe(AU)

Isolation, expansion, differentiation and growth kinetics essay in mesenchymal stem cells culture from the bone marrow of collared peccaries (Tayassu tajacu)

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. [...]

Development of organs and fat deposition in indigenous goat kids under feed restriction / Desenvolvimento dos órgãos e deposição de gorduras em cabritos Canindé sob restrição alimentar

The study aimed to evaluate the effect of feed restriction on the development of organs and fat deposition in castrated Caninde goats. Twenty one goats were distributed in a completely randomized design, with initial body weight of 15.9 ± 1.03kg. The goats were allocated into three levels of feed restriction (seven animals per level): ad libitum; moderate restriction (restriction of 20% on the amount of feed consumed by the animals fed ad libitum) and severe restriction (40% restriction on the amount of feed consumed by the animals fed ad libitum). The diet consisted of 55% forage (Tifton) and 45% concentrate. At 110 days of experiment, the goats were slaughtered with abody weight of 23.5kg ± 2.5kg. The components included the internal organs (spleen, heart, liver, pancreas, lungs, kidneys, blood) the emptied and cleaned digestive tract and fat deposits (in heart, mesenteric, omental and pelvic-renal) were weighed. Feed restriction in Canindé goats affected the weight of organs and fat deposition in regions more involved with the energy storage function (mesenteric, omental and pelvic-renal) (P 0.05), however, feed restriction does not affect the percentage of the organs in relation to EBW (P > 0.05), indicating that even under feed restriction organ development is proportional to the development of the body.

Objetivou-se avaliar o efeito da restrição alimentar sobre o desenvolvimento dos órgãos e deposição de gordura em caprinos Canindé castrados. Foram utilizados 21 cabritos confinados, em um delineamento inteiramente casualizado, com peso inicial de 15,9 ± 1,03kg. Os cabritos foram alocados em três níveis de restrição alimentar (sete animais por nível): ad libitum (alimentados à vontade); restrição moderada (restrição de 20% em relação à quantidade de matéria natural consumida pelos animais alimentados ad libitum) e restrição severa (restrição de 40% em relação à quantidade de ração consumida pelos animais alimentados ad libitum). A ração experimental apresentou uma proporção de 55% de volumoso (Tifton) e 45% de concentrado. Aos 110 dias de experimento os cabritos foram abatidos com peso médio de 23,5 kg ± 2,5 kg. Esvaziou-se o trato gastrointestinal (TGI), a bexiga e a vesícula biliar e foram mensurados os seus pesos para determinação do peso de corpo vazio (PCV). Foram separados e registrados os pesos dos órgãos (baço, coração, fígado, pâncreas, pulmões, rins, sangue, TGI) e dos depósitos de gordura (cardíaca, mesentérica, omental e pélvico-renal). A restrição alimentar em caprinos Canindé afetou o peso absoluto dos órgãos e das gorduras (P 0.05) que estão mais envolvidas com a função de reserva energética (mesentérica, omental e pélvico-renal), no entanto, não afetou o pe

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.(AU)

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.(AU)

Adipose-derived stem cells (ADSC) in the viability of a random pattern dorsal skin flap in rats

To evaluate the viability of random pattern dorsal skin flaps in rats after injection of adipose-derived stem cells (ADSC). Thirty five adult male Wistar EPM rats (weight 250-300 g) were distributed, at random, in two groups. I- Control (flap elevation with injection of saline solution) with fifteen animals and II- Experimental (flap elevation with injection of ADSC ) with fifteen animal. The ADSC were isolated from others five adult male rats. A dorsal skin flap measuring 10x4 cm was raised and a plastic barrier was placed between the flap and its bed in both groups and the injection (cells or saline solution) were perfomed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation to verify their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). CONCLUSIONS: The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random random pattern dorsal skin flaps in rats.(AU)