Resumo
Degenerative diseases, such as osteoporosis, could be treated by stem cells. The aim of this study was to identify the gene expression of bone marrow mesenchymal stem cells (BM-MSC) derived from Sprague Dawley rats and to assess the effect of Cissus quadrangularis Salisb. extract on their maturation into bone cells. The BM-MSC were divided into three groups: (a) BM-MSCs + osteoblast cell growth basal medium as the positive control; (b) BM-MSCs + Dulbecco's modified eagle's medium (DMEM) + 0.3 mg/mL methanol extract of C. quadrangularis as methanol group; and (c) BM-MSC + DMEM + 0.3 mg/mL ethyl acetate extract of C. quadrangularis as ethyl acetate group. A relative quantification approach using was used to analyze the expression of the alp (alkaline phosphatase) gene, with the beta-actin gene was used to normalize the expression of the alp gene. The intra-assay variation was calculated to validate the RT-qPCR data. Our study found that the intra-assay variation value was acceptable, with most of the coefficients of variability (CV) value <5. Ethyl acetate solvent outperformed methanol solvent in extracting the active compound C. quadrangularis. In the ethyl acetate extract group, the expression of the alp gene increased three times compared to the positive control. In methanol extract group, the expression of alp gene was lower six times compared to positive control. This study suggests that C. quadrangularis extracts using ethyl acetate could induce the maturation of BM-MSCs. However, further studies are warrant to confirm this effect using different indicators.
Doenças degenerativas, como a osteoporose, podem ser tratadas por células-tronco. O objetivo deste estudo foi identificar a expressão gênica de células-tronco mesenquimais da medula óssea (BM-MSCs) derivadas de ratos Sprague Dawley, bem como sua proliferação e diferenciação em células ósseas afetadas pelo extrato de Cissus quadrangularis Salisb. As BM-MSCs foram divididas em três grupos: (1) BM-MSCs + meio basal de crescimento de osteoblastos como controle positivo; (2) BM-MSCs + meio de Eagle modificado por Dulbecco (DMEM) + extrato de metanol; e (3) BM-MSCs + DMEM + extrato de acetato de etila de C. quadrangularis. Uma abordagem de quantificação relativa foi usada para analisar a expressão do gene alp (fosfatase alcalina), com o gene da beta-actina sendo usado para normalizar a expressão do gene alp em cada tratamento. A variação intraensaio foi calculada para validar os dados de RT-qPCR. Os resultados mostraram que o valor da variação intraensaio é aceitável, com a maioria dos valores dos coeficientes de variabilidade (CV) < 5. Além disso, no tratamento T2, a expressão do gene alp aumentou três vezes em relação ao controle positivo. No tratamento T1, o gene alp apresentou nível de expressão seis vezes menor em comparação com o controle positivo. Além disso, o solvente acetato de etila superou o solvente metanol na extração do composto ativo C. quadrangularis Salisb. Este estudo demonstra que extratos de C. quadrangularis Salisb., particularmente com acetato de etila como solvente, podem induzir a proliferação e diferenciação de BM-MSCs. Com base na expressão do gene alp, C. quadrangularis tem o potencial de corrigir a perda óssea.
Assuntos
Animais , Ratos , Osteoporose , Extratos Vegetais , Cissus , Células-Tronco MesenquimaisResumo
Infertility is one of the most prevalent health disorders in reproductive-age males and females. Ficus carica (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dosedependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.(AU)
Assuntos
Animais , Camundongos , Extratos Vegetais/efeitos adversos , Ficus/efeitos adversos , Camundongos/embriologia , Citotoxicidade Imunológica , Células-Tronco Germinativas Adultas/citologiaResumo
A cinomose canina é uma doença infectocontagiosa causada pelo vírus Paramyxoviridae, que acomete cães de todas as idades, entretanto principalmente cães filhotes não vacinados. Uma das sequelas da doença é neurológica que, por muitas vezes, é de difícil tratamento. A terapia com células-tronco é uma alternativa para atenuar os sinais e sequelas neurológicas, propiciando melhor qualidade de vida para o animal. As células-tronco são células com alta capacidade de auto renovação e diferenciação, e, portanto, uma alternativa de tratamento para as sequelas neurológicas derivada pela cinomose.
Canine distemper is an infectocontagious disease caused by Paramyxoviridae virus, which affects dogs of all ages, however mainly unvaccinated puppies. Neurological manifestation are common in canine distemper which is often difficult to treat. Stem cell therapy is an alternative to attenuate neurological signs and sequel, providing better quality of life for the animal. Stem cells have high capacity for self-renewal and differentiation, and an alternative treatment for neurological sequel derived by distemper.
Assuntos
Animais , Cães , Transplante de Células-Tronco/veterinária , Cinomose/terapia , Células-Tronco , CãesResumo
The objective of this study was to investigate the in vitro action of triiodothyronine (T3) on the chondrogenic differentiation of adipose tissue-derived stem cells (ASCs) of female rats, with different time periods and doses. ASCs were extracted from female Wistar rats and were cultured in chondrogenic medium with and without the presence of T3. Five groups were established: 1) ASCs without T3; and 2,3,4,5) ASCs with 0.01, 1, 100 and 1,000 nM T3, respectively). After 7, 14 and 21 days, cell morphology, chondrogenic matrix formation, and expression of Sox9, aggrecan, collagen II, and collagen X were evaluated. The Student-Newman-Keuls test was used. ASCs showed CD54, CD73, and CD90 before chondrogenic differentiation. The hormone treatment did not alter chondrogenic matrix formation, Sox9 expression at 14 or 21 days, or expression of collagen II or collagen X at any time. However, the 0.01, 1, and 1000 nM T3 doses decreased Sox9 expression at 7 days. In conclusion, chondrogenic differentiation of ASCs of female rats is not influenced by T3.
O objetivo do presente trabalho foi verificar o efeito in vitro da triiodotironina (T3) na diferenciação condrogênica de células tronco mesenquimais do tecido adiposo (CTM-TA) de ratas, durante vários períodos e em várias doses. CTM-TA foram coletadas de ratas Wistar e cultivadas em meio condrogênico com ou sem a presença de T3. Constitui-se cinco grupos: 1) CTM-TA sem T3; e 2,3,4,5) CTM-TA com T3 (0,01; 1; 100 e 1000 nM, respectivamente). Após sete, 14 e 21 dias, foram avaliados morfologia celular, formação de matriz condrogênica e expressão de Sox9, agrecano, colágeno II e colágeno X. Para as análises foi utilizado o teste de Student Newman Keuls. CTM-TA expressaram CD54, CD73 e CD90 antes da diferenciação condrogênica. O tratamento hormonal não alterou a formação de matriz condrogênica e a expressão de Sox9 aos 14 e 21 dias e expressão dos colágenos II e X em nenhum dos períodos avaliados. No entanto, as doses de 0,01; 1 e 1000 nM T3 diminuíram a expressão de Sox9 aos 7 dias. Conclui-se que a diferenciação condrogênica de CTM-TA de ratas não é influenciada pela T3.
Assuntos
Animais , Feminino , Ratos , Células-Tronco/fisiologia , Tri-Iodotironina/análise , Tecido Adiposo/fisiologia , Condrogênese/fisiologiaResumo
Abstract The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.
Resumo
The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.(AU)
Assuntos
Animais , Cães , Cães , Células-Tronco Hematopoéticas/classificação , Saco Vitelino , Sistema HematopoéticoResumo
Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.
Assuntos
Animais , Células-Tronco Mesenquimais , Puma/anatomia & histologia , Puma/genética , Tecido AdiposoResumo
Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.(AU)
Assuntos
Animais , Puma/anatomia & histologia , Puma/genética , Células-Tronco Mesenquimais , Tecido AdiposoResumo
A criocirurgia tem sido utilizada no tratamento de diferentes enfermidades de sistemas e órgãos. Contudo, são relatados efeitos adversos, como cicatrização lenta, cicatrizes extensas, disfunção estética e funcional. As lesões que ocorrem naturalmente pela exposição ao frio extremo, comumente, resultam em gangrena. O presente trabalho teve como objetivo avaliar a influência das células-tronco mesenquimais de origem adiposa (ADSCs) na fase de proliferação da cicatrização de feridas cutâneas. Por meio da aplicação do nitrogênio líquido pela técnica do spray aberto, realizou-se a indução de uma ferida, de aproximadamente 15mm de diâmetro, na região dorsal de cada rato. A ferida recebeu o tratamento de acordo com o grupo ao qual pertencia: 1) aplicação das ADSCs no 15º dia (grupo tratado); 2) aplicação da solução cloreto de sódio 0,9% no 15º dia (grupo sham); 3) nenhuma intervenção até o momento da eutanásia (grupo controle). O grupo tratado com as ADSCs apresentou as maiores taxas de contração média das feridas e obteve diferença estatisticamente significativa em relação ao grupo sham quanto à neovascularização. A terapia com as ADSCs proporcionou uma relevante evolução clínica das feridas, podendo ser constatada ao final do período de avaliação por cicatrizes mais estreitas e compridas.(AU)
Cryosurgery has been used to treat different diseases of systems and organs, although adverse effects have been reported such as delayed wound healing, large scars, esthetical deformation and functional impairment. Injuries caused naturally by the exposure to extreme cold weather conditions mostly result in gangrene. This study aims to evaluate the influence of adipose-derived stem cells (ADSCs) in the proliferation phase on cutaneous wound healing. Through the application of liquid nitrogen by the spraying technique, a 15 millimeter diameter lesion was produced in the dorsal region of each rat. The wound received treatment according to the group it belonged: 1) ADSCs application on the 15th day (treated group); 2) application of 0.9% sodium chloride solution on the 15th day (sham group); 3) no intervention until euthanasia (control group). The group treated with ADSCs showed the highest wound average contraction rate; this group got a significant statistical difference in relation to the sham group when it refers to neovascularization. The ADSCs therapy provides an important clinical evolution of wounds. This was verified at the end of the evaluation period through narrower and longer scars.(AU)
Assuntos
Animais , Ratos , Cicatrização/fisiologia , Queimaduras/veterinária , Criocirurgia/veterinária , Células-Tronco Mesenquimais , Ferimentos e Lesões/terapia , Ratos Wistar , Agentes de Resfriamento , Resposta ao Choque FrioResumo
A criocirurgia tem sido utilizada no tratamento de diferentes enfermidades de sistemas e órgãos. Contudo, são relatados efeitos adversos, como cicatrização lenta, cicatrizes extensas, disfunção estética e funcional. As lesões que ocorrem naturalmente pela exposição ao frio extremo, comumente, resultam em gangrena. O presente trabalho teve como objetivo avaliar a influência das células-tronco mesenquimais de origem adiposa (ADSCs) na fase de proliferação da cicatrização de feridas cutâneas. Por meio da aplicação do nitrogênio líquido pela técnica do spray aberto, realizou-se a indução de uma ferida, de aproximadamente 15mm de diâmetro, na região dorsal de cada rato. A ferida recebeu o tratamento de acordo com o grupo ao qual pertencia: 1) aplicação das ADSCs no 15º dia (grupo tratado); 2) aplicação da solução cloreto de sódio 0,9% no 15º dia (grupo sham); 3) nenhuma intervenção até o momento da eutanásia (grupo controle). O grupo tratado com as ADSCs apresentou as maiores taxas de contração média das feridas e obteve diferença estatisticamente significativa em relação ao grupo sham quanto à neovascularização. A terapia com as ADSCs proporcionou uma relevante evolução clínica das feridas, podendo ser constatada ao final do período de avaliação por cicatrizes mais estreitas e compridas.(AU)
Cryosurgery has been used to treat different diseases of systems and organs, although adverse effects have been reported such as delayed wound healing, large scars, esthetical deformation and functional impairment. Injuries caused naturally by the exposure to extreme cold weather conditions mostly result in gangrene. This study aims to evaluate the influence of adipose-derived stem cells (ADSCs) in the proliferation phase on cutaneous wound healing. Through the application of liquid nitrogen by the spraying technique, a 15 millimeter diameter lesion was produced in the dorsal region of each rat. The wound received treatment according to the group it belonged: 1) ADSCs application on the 15th day (treated group); 2) application of 0.9% sodium chloride solution on the 15th day (sham group); 3) no intervention until euthanasia (control group). The group treated with ADSCs showed the highest wound average contraction rate; this group got a significant statistical difference in relation to the sham group when it refers to neovascularization. The ADSCs therapy provides an important clinical evolution of wounds. This was verified at the end of the evaluation period through narrower and longer scars.(AU)
Assuntos
Animais , Ratos , Cicatrização/fisiologia , Queimaduras/veterinária , Criocirurgia/veterinária , Células-Tronco Mesenquimais , Ferimentos e Lesões/terapia , Ratos Wistar , Agentes de Resfriamento , Resposta ao Choque FrioResumo
This study aimed to histologically evaluate the quality of tissue repair in equine suspensory ligament treated with two cell therapy protocols. All four limbs of six animals were operated simultaneously to remove a fragment in each ligament using a skin biopsy punch. Two days later, intralesional injections were performed using bone marrow mononuclear fraction (BM group), cultivated cells derived from adipose tissue (AT group), saline (positive control group), or no treatment (negative control group), in such way that each horse received all treatments. After sixty days biopsies were performed for histological analysis (H & E, Masson's trichrome and picrosirius red) and immunohistochemistry analysis (collagen type III). Histological findings (H & E and Masson's trichrome), birefringence intensity (through picrosirius) and collagen type III expression (through immunohistochemistry) were analyzed. Samples from treated groups had better birefringence intensity (P=0.007) and fiber alignment scores were superior compared to controls, though not statistically significant (P=0.08). Presence of inflammatory cells and intense staining for collagen type III occurred in all groups demonstrating an active healing process. In conclusion, both protocols resulted in improvement of tissue repair indicating their potential to be used as an adjuvant treatment of equine suspensory ligament disorders.(AU)
Este estudo teve como objetivo a avaliação histológica e imunoistoquímica do reparo do ligamento suspensório equino tratado com dois protocolos de terapia celular. Os quatro membros dos seis animais do experimento foram submetidos a procedimento cirúrgico em que um fragmento de cada ligamento foi retirado, utilizando-se punch de biópsia. Dois dias após o procedimento, aplicações intralesionais foram realizadas, por meio de aspirado de medula óssea (bone marrow-BM), células mesenquimais derivadas de tecido adiposo (adipose tissue-AT), solução salina (positive control group-PC) ou controle (negative control-NC). Após 60 dias, biópsias foram retiradas da região de reparo dos ligamentos e foram submetidas à análise histológica (HE, tricrômio de Masson, picrosírius red) e imunoistoquímica (colágeno tipo III). Diferentes variáveis histológicas (HE e tricrômio de Masson), a intensidade de birrefringência das fibras colágenas (picrosírius red) e a expressão de colágeno tipo III foram avaliadas. Os grupos tratados apresentaram maior birrefringência (P=0,007) e alinhamento de fibras (P=0,08) comparados ao controle, para o qual o resultado não se mostrou estatisticamente significativo. Achados histológicos e imunoistoquímicos demonstraram um processo ativo de reparo tecidual em todos os grupos. Concluiu-se que os dois protocolos de terapia celular apresentaram melhora no reparo tecidual, demonstrando potencial terapêutico adjuvante no tratamento de afecções do ligamento suspensório equino.(AU)
Assuntos
Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/veterinária , Cavalos/anatomia & histologia , Ligamentos/anatomia & histologia , Ligamentos/química , Imuno-Histoquímica/veterináriaResumo
This study aimed to histologically evaluate the quality of tissue repair in equine suspensory ligament treated with two cell therapy protocols. All four limbs of six animals were operated simultaneously to remove a fragment in each ligament using a skin biopsy punch. Two days later, intralesional injections were performed using bone marrow mononuclear fraction (BM group), cultivated cells derived from adipose tissue (AT group), saline (positive control group), or no treatment (negative control group), in such way that each horse received all treatments. After sixty days biopsies were performed for histological analysis (H & E, Masson's trichrome and picrosirius red) and immunohistochemistry analysis (collagen type III). Histological findings (H & E and Masson's trichrome), birefringence intensity (through picrosirius) and collagen type III expression (through immunohistochemistry) were analyzed. Samples from treated groups had better birefringence intensity (P=0.007) and fiber alignment scores were superior compared to controls, though not statistically significant (P=0.08). Presence of inflammatory cells and intense staining for collagen type III occurred in all groups demonstrating an active healing process. In conclusion, both protocols resulted in improvement of tissue repair indicating their potential to be used as an adjuvant treatment of equine suspensory ligament disorders.(AU)
Este estudo teve como objetivo a avaliação histológica e imunoistoquímica do reparo do ligamento suspensório equino tratado com dois protocolos de terapia celular. Os quatro membros dos seis animais do experimento foram submetidos a procedimento cirúrgico em que um fragmento de cada ligamento foi retirado, utilizando-se punch de biópsia. Dois dias após o procedimento, aplicações intralesionais foram realizadas, por meio de aspirado de medula óssea (bone marrow-BM), células mesenquimais derivadas de tecido adiposo (adipose tissue-AT), solução salina (positive control group-PC) ou controle (negative control-NC). Após 60 dias, biópsias foram retiradas da região de reparo dos ligamentos e foram submetidas à análise histológica (HE, tricrômio de Masson, picrosírius red) e imunoistoquímica (colágeno tipo III). Diferentes variáveis histológicas (HE e tricrômio de Masson), a intensidade de birrefringência das fibras colágenas (picrosírius red) e a expressão de colágeno tipo III foram avaliadas. Os grupos tratados apresentaram maior birrefringência (P=0,007) e alinhamento de fibras (P=0,08) comparados ao controle, para o qual o resultado não se mostrou estatisticamente significativo. Achados histológicos e imunoistoquímicos demonstraram um processo ativo de reparo tecidual em todos os grupos. Concluiu-se que os dois protocolos de terapia celular apresentaram melhora no reparo tecidual, demonstrando potencial terapêutico adjuvante no tratamento de afecções do ligamento suspensório equino.(AU)
Assuntos
Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/veterinária , Cavalos/anatomia & histologia , Ligamentos/anatomia & histologia , Ligamentos/química , Imuno-Histoquímica/veterináriaResumo
The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)
O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)
Assuntos
Animais , Células-Tronco/classificação , Tecido Adiposo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/análise , Células GerminativasResumo
The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)
O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)
Assuntos
Animais , Tecido Adiposo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/análise , Células-Tronco/classificação , Células GerminativasResumo
The present study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) infusion, derived from adipose tissue, on reduction of local and remote tissue damage caused by the event of experimental intestinal I/R in New Zealand breed rabbits. For obtaining, characterization, and cultivation of MSC derived from adipose tissue (MSC-Adp), 3 juvenile animals (four months old) were used. The cells were considered to be viable for therapy after the fourth passage (in vitro phase). For the in vivo stage, 24 young adult animals (six months old) were used, weighing approximately 3.5 kg, in which were randomly divided into two groups, called: IR treated with MSC (I2H/R5H MSC 3D; I2H/R5H MSC 7D); IR treated with PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). The animals were anesthetized and submitted to pre-retro-umbilical midline celiotomy. The extramural peri-intestinal marginal artery was located and clamped (predetermined and standardized region) with the aid of a vascular clip, promoting a 2 hour blood flow interruption. After this period, blood flow was reestablished, inhalatory anesthesia was suspended, and the animals awaken. After 5 hours of reperfusion, the treatments were performed by intravenous infusion according to the experimental groups. The animals were evaluated 72 hours and seven days after the treatment as for the macroscopic appearance (color and peristaltism) of the jejunal segment, and by histological evaluation of the ischemic segment for the presence or absence of destruction of the intestinal mucosa, edema, bleeding, dilation of lymph vessels, and presence of polymorphonuclear inflammatory cells, both in the mucosa and submucosa. The observed results revealed that the groups treated with MSC-Adp obtained smaller mucosal and submucosal lesions when compared to the groups treated with PBS. Also, MSC-Adp treated groups obtained controlled inflammatory response and higher mitotic rate, outcomes related to the therapeutic potential of MSC. Infusion of stem cells attenuated the lesions caused by intestinal I/R in both MSC groups when compared to the group treated with PBS.(AU)
O presente estudo teve como principal objetivo avaliar a eficácia da infusão células tronco mesenquimais (CTM) derivada de tecido adiposo sobre diminuição das lesões teciduais locais e remotas, causadas pelo evento de I/R intestinal experimental, em coelhos da raça Nova Zelândia. Para obtenção, cultivo e caracterização das CTM provenientes de tecido adiposo (ADCTM) foram utilizados 3 animais jovens. As células foram consideradas viáveis para terapia a partir da quarta passagem (fase in vitro). Para etapa in vivo foram utilizados 24 animais, adulto-jovens, pesando aproximadamente 3,5kg, divididos aleatoriamente em dois grupos experimentais, denominados IR Tratado com CTM (I2H/R5H CTM 3D; I2H/R5H CTM 7D); IR Tratado PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). Os animais foram anestesiados e submetidos à celiotomia mediana pré-retroumbilical. A artéria marginal peri-intestinal extramural foi localizada e clampeada (região predeterminada e padronizada) com auxílio de um clipe vascular, promovendo uma interrupção do fluxo sanguíneo durante 2 horas. Após esse período, o fluxo sanguíneo foi restabelecido, a anestesia inalatória suspendida e os animais despertados. Após 5 horas de reperfusão realizou-se os tratamentos por infusão endovenosa, conforme grupos experimentais. Os animais foram avaliados 72 horas e sete dias após o tratamento quanto ao aspecto macroscópico (coloração e peristaltismo) do segmento jejunal e por meio de avaliação histológica do segmento isquemiado quanto à presença ou ausência de destruição de mucosa intestinal, edema, hemorragia, dilatação de vasos linfáticos e presença de células inflamatórias polimorfornucleares, tanto em mucosa quanto submucosa. Os resultados observados revelaram que os grupos tratados com ADCTM obtiveram menores lesões em mucosa e submucosa quando comprados aos grupos tratados com PBS. Ainda os grupos tratados com ADCTM obtiveram resposta inflamatória controlada e maior taxa mitótica, resultados relacionados ao potencial terapêutico das CTM.(AU)
Assuntos
Animais , Coelhos , Coelhos/anatomia & histologia , Coelhos/genética , Coelhos/lesões , Infusões Intravenosas/veterinária , Transplante de Células-Tronco Mesenquimais/estatística & dados numéricos , Isquemia/veterináriaResumo
The present study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) infusion, derived from adipose tissue, on reduction of local and remote tissue damage caused by the event of experimental intestinal I/R in New Zealand breed rabbits. For obtaining, characterization, and cultivation of MSC derived from adipose tissue (MSC-Adp), 3 juvenile animals (four months old) were used. The cells were considered to be viable for therapy after the fourth passage (in vitro phase). For the in vivo stage, 24 young adult animals (six months old) were used, weighing approximately 3.5 kg, in which were randomly divided into two groups, called: IR treated with MSC (I2H/R5H MSC 3D; I2H/R5H MSC 7D); IR treated with PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). The animals were anesthetized and submitted to pre-retro-umbilical midline celiotomy. The extramural peri-intestinal marginal artery was located and clamped (predetermined and standardized region) with the aid of a vascular clip, promoting a 2 hour blood flow interruption. After this period, blood flow was reestablished, inhalatory anesthesia was suspended, and the animals awaken. After 5 hours of reperfusion, the treatments were performed by intravenous infusion according to the experimental groups. The animals were evaluated 72 hours and seven days after the treatment as for the macroscopic appearance (color and peristaltism) of the jejunal segment, and by histological evaluation of the ischemic segment for the presence or absence of destruction of the intestinal mucosa, edema, bleeding, dilation of lymph vessels, and presence of polymorphonuclear inflammatory cells, both in the mucosa and submucosa. The observed results revealed that the groups treated with MSC-Adp obtained smaller mucosal and submucosal lesions when compared to the groups treated with PBS...(AU)
O presente estudo teve como principal objetivo avaliar a eficácia da infusão células tronco mesenquimais (CTM) derivada de tecido adiposo sobre diminuição das lesões teciduais locais e remotas, causadas pelo evento de I/R intestinal experimental, em coelhos da raça Nova Zelândia. Para obtenção, cultivo e caracterização das CTM provenientes de tecido adiposo (ADCTM) foram utilizados 3 animais jovens. As células foram consideradas viáveis para terapia a partir da quarta passagem (fase in vitro). Para etapa in vivo foram utilizados 24 animais, adulto-jovens, pesando aproximadamente 3,5kg, divididos aleatoriamente em dois grupos experimentais, denominados IR Tratado com CTM (I2H/R5H CTM 3D; I2H/R5H CTM 7D); IR Tratado PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). Os animais foram anestesiados e submetidos à celiotomia mediana pré-retroumbilical. A artéria marginal peri-intestinal extramural foi localizada e clampeada (região predeterminada e padronizada) com auxílio de um clipe vascular, promovendo uma interrupção do fluxo sanguíneo durante 2 horas. Após esse período, o fluxo sanguíneo foi restabelecido, a anestesia inalatória suspendida e os animais despertados. Após 5 horas de reperfusão realizou-se os tratamentos por infusão endovenosa, conforme grupos experimentais. Os animais foram avaliados 72 horas e sete dias após o tratamento quanto ao aspecto macroscópico (coloração e peristaltismo) do segmento jejunal e por meio de avaliação histológica do segmento isquemiado quanto à presença ou ausência de destruição de mucosa intestinal, edema, hemorragia, dilatação de vasos linfáticos e presença de células inflamatórias polimorfornucleares, tanto em mucosa quanto submucosa. Os resultados observados revelaram que os grupos tratados com ADCTM obtiveram menores lesões em mucosa e submucosa quando comprados aos grupos tratados com PBS...(AU)
Assuntos
Animais , Coelhos , Coelhos/anatomia & histologia , Coelhos/genética , Coelhos/lesões , Infusões Intravenosas/veterinária , Transplante de Células-Tronco Mesenquimais , Isquemia/veterináriaResumo
ABSTRACT: The present study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) infusion, derived from adipose tissue, on reduction of local and remote tissue damage caused by the event of experimental intestinal I/R in New Zealand breed rabbits. For obtaining, characterization, and cultivation of MSC derived from adipose tissue (MSC-Adp), 3 juvenile animals (four months old) were used. The cells were considered to be viable for therapy after the fourth passage (in vitro phase). For the in vivo stage, 24 young adult animals (six months old) were used, weighing approximately 3.5 kg, in which were randomly divided into two groups, called: IR treated with MSC (I2H/R5H MSC 3D; I2H/R5H MSC 7D); IR treated with PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). The animals were anesthetized and submitted to pre-retro-umbilical midline celiotomy. The extramural peri-intestinal marginal artery was located and clamped (predetermined and standardized region) with the aid of a vascular clip, promoting a 2 hour blood flow interruption. After this period, blood flow was reestablished, inhalatory anesthesia was suspended, and the animals awaken. After 5 hours of reperfusion, the treatments were performed by intravenous infusion according to the experimental groups. The animals were evaluated 72 hours and seven days after the treatment as for the macroscopic appearance (color and peristaltism) of the jejunal segment, and by histological evaluation of the ischemic segment for the presence or absence of destruction of the intestinal mucosa, edema, bleeding, dilation of lymph vessels, and presence of polymorphonuclear inflammatory cells, both in the mucosa and submucosa. The observed results revealed that the groups treated with MSC-Adp obtained smaller mucosal and submucosal lesions when compared to the groups treated with PBS. Also, MSC-Adp treated groups obtained controlled inflammatory response and higher mitotic rate, outcomes related to the therapeutic potential of MSC. Infusion of stem cells attenuated the lesions caused by intestinal I/R in both MSC groups when compared to the group treated with PBS.
RESUMO: O presente estudo teve como principal objetivo avaliar a eficácia da infusão células tronco mesenquimais (CTM) derivada de tecido adiposo sobre diminuição das lesões teciduais locais e remotas, causadas pelo evento de I/R intestinal experimental, em coelhos da raça Nova Zelândia. Para obtenção, cultivo e caracterização das CTM provenientes de tecido adiposo (ADCTM) foram utilizados 3 animais jovens. As células foram consideradas viáveis para terapia a partir da quarta passagem (fase in vitro). Para etapa in vivo foram utilizados 24 animais, adulto-jovens, pesando aproximadamente 3,5kg, divididos aleatoriamente em dois grupos experimentais, denominados IR Tratado com CTM (I2H/R5H CTM 3D; I2H/R5H CTM 7D); IR Tratado PBS (I2H/R5H PBS 3D; I2H/R5H PBS 7D). Os animais foram anestesiados e submetidos à celiotomia mediana pré-retroumbilical. A artéria marginal peri-intestinal extramural foi localizada e clampeada (região predeterminada e padronizada) com auxílio de um clipe vascular, promovendo uma interrupção do fluxo sanguíneo durante 2 horas. Após esse período, o fluxo sanguíneo foi restabelecido, a anestesia inalatória suspendida e os animais despertados. Após 5 horas de reperfusão realizou-se os tratamentos por infusão endovenosa, conforme grupos experimentais. Os animais foram avaliados 72 horas e sete dias após o tratamento quanto ao aspecto macroscópico (coloração e peristaltismo) do segmento jejunal e por meio de avaliação histológica do segmento isquemiado quanto à presença ou ausência de destruição de mucosa intestinal, edema, hemorragia, dilatação de vasos linfáticos e presença de células inflamatórias polimorfornucleares, tanto em mucosa quanto submucosa. Os resultados observados revelaram que os grupos tratados com ADCTM obtiveram menores lesões em mucosa e submucosa quando comprados aos grupos tratados com PBS. Ainda os grupos tratados com ADCTM obtiveram resposta inflamatória controlada e maior taxa mitótica, resultados relacionados ao potencial terapêutico das CTM.
Resumo
Adipose tissue-derived stem cells (ADSCs) are an attractive source of mesenchymal stem cells (MSCs) for use in tissue engineering and clinical applications. This paper focuses on the characterization of ADSCs used as immunosuppressive agent in rabbits undergoing partial allograft for urine bladder restorage. For this study highlighted the characterization of the ADSCs used as immunosuppressive agents in rabbits submitted to partial allograft for restoration of the urinary vesicle, using 25 animals, six months old, New Zealand. ADSCs at the third peal were characterized by the MSC-specific CD105, CD73 and CD90 expression and by the absence of the hematopoietic marker CD45, as revealed by flow cytometry analysis. Moreover, ADSCs were efficient in preventing allograft rejection from the urinary bladder, as judged by biochemical, clinical and ultrasonography analysis. Together, these results compose characterization of protein expression profiles and immunosuppressive functionality of ADSCs in rabbits, which had undergone partial allografts of the urinary bladder, foreseeing future applications in clinical practice.(AU)
As células mesenquimais derivadas de tecido adiposo (ADSCs) são uma fonte atraente de células-tronco mesenquimais (MSCs) para uso na engenharia de tecidos e suas aplicações clínicas. Este trabalho destacou a caracterização das ADSCs utilizadas como agentes imunossupressores em coelhos submetidos a aloenxerto parcial para restauração da vesícula urinária, sendo utilizados 25 animais, de seis meses de idade, Nova Zelândia. As ADSCs, após o terceiro repique, foram caracterizadas pela expressão específica de MSC CD105, CD73 e CD90 e pela ausência do marcador hematopoiético CD45, tal como revelado por análise de citometria de fluxo. Além disso, os ADSCs foram eficientes na prevenção da rejeição de aloenxertos da vesícula urinária, conforme avaliado por análises clínica, bioquímica e ultrassonográfica. Juntos, esses resultados compõem a caracterização dos perfis de expressão proteica e a funcionalidade imunossupressora de ADSCs em coelhos, que sofreram aloenxertos parciais da bexiga, prevendo futuras aplicações na prática clínica.(AU)
Assuntos
Animais , Coelhos , Coelhos , Bexiga Urinária/transplante , Aloenxertos/citologia , Terapia Baseada em Transplante de Células e Tecidos/veterinária , Imunossupressores , Citometria de Fluxo/veterináriaResumo
Adipose tissue-derived stem cells (ADSCs) are an attractive source of mesenchymal stem cells (MSCs) for use in tissue engineering and clinical applications. This paper focuses on the characterization of ADSCs used as immunosuppressive agent in rabbits undergoing partial allograft for urine bladder restorage. For this study highlighted the characterization of the ADSCs used as immunosuppressive agents in rabbits submitted to partial allograft for restoration of the urinary vesicle, using 25 animals, six months old, New Zealand. ADSCs at the third peal were characterized by the MSC-specific CD105, CD73 and CD90 expression and by the absence of the hematopoietic marker CD45, as revealed by flow cytometry analysis. Moreover, ADSCs were efficient in preventing allograft rejection from the urinary bladder, as judged by biochemical, clinical and ultrasonography analysis. Together, these results compose characterization of protein expression profiles and immunosuppressive functionality of ADSCs in rabbits, which had undergone partial allografts of the urinary bladder, foreseeing future applications in clinical practice.(AU)
As células mesenquimais derivadas de tecido adiposo (ADSCs) são uma fonte atraente de células-tronco mesenquimais (MSCs) para uso na engenharia de tecidos e suas aplicações clínicas. Este trabalho destacou a caracterização das ADSCs utilizadas como agentes imunossupressores em coelhos submetidos a aloenxerto parcial para restauração da vesícula urinária, sendo utilizados 25 animais, de seis meses de idade, Nova Zelândia. As ADSCs, após o terceiro repique, foram caracterizadas pela expressão específica de MSC CD105, CD73 e CD90 e pela ausência do marcador hematopoiético CD45, tal como revelado por análise de citometria de fluxo. Além disso, os ADSCs foram eficientes na prevenção da rejeição de aloenxertos da vesícula urinária, conforme avaliado por análises clínica, bioquímica e ultrassonográfica. Juntos, esses resultados compõem a caracterização dos perfis de expressão proteica e a funcionalidade imunossupressora de ADSCs em coelhos, que sofreram aloenxertos parciais da bexiga, prevendo futuras aplicações na prática clínica.(AU)
Assuntos
Animais , Coelhos , Coelhos , Bexiga Urinária/transplante , Aloenxertos/citologia , Terapia Baseada em Transplante de Células e Tecidos/veterinária , Imunossupressores , Citometria de Fluxo/veterináriaResumo
Purpose: To evaluate the effects of mesenchymal stem cells on liver regeneration in rats following a 70% hepatectomy. Methods: Forty rats were subjected to 70% hepatectomy and then ~106 mesenchymal stem cells (test group), or saline solution (control group), were infused into their livers via the portal vein. Each treatment group was divided into early and late subgroups (euthanized 3 d and 5 d following the operation, respectively). Group comparisons of Albumin, aminotransaminases (AST, ALT), and Alcaline Phosphatase (AP) levels, proliferative index (ki-67+ straining), and mitotic cell counts were conducted. Results: No significant differences in liver regeneration rate, number of mitoses, proliferative index, or serum levels of albumin, AST, or AP were observed. ALT levels were higher in the test group than in the control group (p .05). Conclusions: Mesenchymal stem-cell therapy did not improve liver regeneration rate 3 d or 5 d after 70% hepatectomy in rats. Likewise, the therapy appeared not to affect liver function, proliferative index, or number of mitoses significantly.(AU)