Structural plasticity and isolation of umbilical cord progenitor cells of agouti (Dasyprocta prymnolopha) raised in captivity / Plasticidade estrutural e isolamento de células progenitoras do cordão umbilical de cutias (Dasyprocta prymnolopha) criadas em cativeiro

The agouti has been used as an experimental model in several studies focused on reproductive biology. The umbilical cord, an embryonic attachment that connects the foetus to the placenta, has been reported as an important anatomical site for obtaining stem cells. The objective of this study was to describe macro- and microscopically the umbilical cord of agoutis at different stages of gestation, to expand and cultivate in vitro the progenitor cells and to report their morphological characteristics. Seven cutias were submitted to caesarean section to collect the umbilical cords: five were destined for studies of cord structure in different stages of gestation (30, 35, 50, 75 and 100 days postcoital), and two were collected in the third stage of gestation for isolation and cell culture. The umbilical cord of cutias assumes a spiral arrangement, with veins and arteries on it starting 50 days after coitus. The arteries present an outer layer of smooth muscle fibres in a longitudinal and circular arrangement and a medium layer of smooth muscle fibres with only longitudinal and intimate orientation and coated by the endothelium. The veins consist of longitudinal smooth muscle fibres with an extract of smooth muscle cells, and the endothelium, in all analysed gestational phases, is a structure bounded by simple pavement epithelial tissue originating from the amnion, adhered to Whartons Jelly and forming the umbilical vessels and allantoid duct. The proposed protocol allowed the collection of a high cellular concentration of umbilical cord progenitor cells from viable cutias.(AU)

A cutia vem sendo utilizada como modelo experimental em diversos estudos voltados à biologia reprodutiva. O cordão umbilical, anexo embrionário que une o feto à placenta, tem sido relatado como um importante sítio anatômico para obtenção de células-tronco. O objetivo deste estudo foi descrever macro e microscopicamente o cordão umbilical de cutias, em fases diferentes da gestação, expandir e cultivar in vitro as células progenitoras e relatar suas características morfológicas. Foram utilizadas sete cutias submetidas à cesariana para a coleta dos cordões umbilicais, cinco foram destinadas aos estudos da estrutura do cordão, em diferentes estágios de gestação (30, 35, 50, 75 e 100 dias pós-coito), e duas, no terço final da gestação, para isolamento e cultivo celular. O cordão umbilical de cutia assume disposição espiralada, com veias e artérias sobre ele a partir dos 50 dias após o coito. As artérias apresentam camada externa de fibras musculares lisas, disposição longitudinal e circular, camada média de fibras musculares lisas, apenas com disposição longitudinal e íntima revestida pelo endotélio. As veias constituídas por fibras musculares lisas longitudinais com um extrato de células musculares lisas e pelo endotélio. Em todas as fases gestacionais analisadas é uma estrutura delimitada por tecido epitelial simples pavimentoso, proveniente do âmnio, aderido a Geleia de Wharton e com formação de vasos umbilicais e ducto alantóide. O protocolo proposto permitiu a coleta de células progenitoras do cordão umbilical de cutias, viáveis com elevada concentração celular.(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)

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. [...]

Isolation, culture and differentiation potential of collared peccary (Tayassu tajacu) adipose-derived stem cells

Background: The understanding of cell biology and the isolation of mesenchymal stem cells in wild animals show prospects for conducting pre-clinical trials in these unconventional animals. The collared peccary (Tayassu tajacu) are suiforms that belong to the Artiodáctyla order, Tayassuidae family and Tayassu genus. They adapt easily to captivity conditions that favors their commercial rearing and is an alternative for biodiversity conservation. To evaluate the collared peccary (Tayassu tajacu) as a potential animal model for the isolation of mesenchymal progenitor cells, cell culture and cell differentiation protocols. Materials, Methods & Results: To perform this research we used four collared peccaries (Tayassu tajacu) from the Nucleus of Study and Preservation of Wild Animals (IBAMA/PI No . 02/08-618) from Federal University of Piauí (UFPI). Adipose tissue fragments were collected from the dorsocervical region and dissociated mechanically in laboratory. The material was placed in an incubator containing CO2 - 95% at 37C and the cultures were expanded to fifth passage, evalluating cell concentration and viability. The culture medium alfa-MEM supplemented was changed every three days. The cell kinetics was evaluated in triplicate using growth curve performed during ten days, plating the initial concentration of 5 x 104 cells/mL per well in P3 six-well culture plate...(AU)

Isolation, culture and differentiation potential of collared peccary (Tayassu tajacu) adipose-derived stem cells

Background: The understanding of cell biology and the isolation of mesenchymal stem cells in wild animals show prospects for conducting pre-clinical trials in these unconventional animals. The collared peccary (Tayassu tajacu) are suiforms that belong to the Artiodáctyla order, Tayassuidae family and Tayassu genus. They adapt easily to captivity conditions that favors their commercial rearing and is an alternative for biodiversity conservation. To evaluate the collared peccary (Tayassu tajacu) as a potential animal model for the isolation of mesenchymal progenitor cells, cell culture and cell differentiation protocols. Materials, Methods & Results: To perform this research we used four collared peccaries (Tayassu tajacu) from the Nucleus of Study and Preservation of Wild Animals (IBAMA/PI No . 02/08-618) from Federal University of Piauí (UFPI). Adipose tissue fragments were collected from the dorsocervical region and dissociated mechanically in laboratory. The material was placed in an incubator containing CO2 - 95% at 37C and the cultures were expanded to fifth passage, evalluating cell concentration and viability. The culture medium alfa-MEM supplemented was changed every three days. The cell kinetics was evaluated in triplicate using growth curve performed during ten days, plating the initial concentration of 5 x 104 cells/mL per well in P3 six-well culture plate...

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil

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 106 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.(AU)

Structural plasticity and isolation of umbilical cord progenitor cells of agouti (Dasyprocta prymnolopha) raised in captivity / Plasticidade estrutural e isolamento de células progenitoras do cordão umbilical de cutias (Dasyprocta prymnolopha) criadas em cativeiro

The agouti has been used as an experimental model in several studies focused on reproductive biology. The umbilical cord, an embryonic attachment that connects the foetus to the placenta, has been reported as an important anatomical site for obtaining stem cells. The objective of this study was to describe macro- and microscopically the umbilical cord of agoutis at different stages of gestation, to expand and cultivate in vitro the progenitor cells and to report their morphological characteristics. Seven cutias were submitted to caesarean section to collect the umbilical cords: five were destined for studies of cord structure in different stages of gestation (30, 35, 50, 75 and 100 days postcoital), and two were collected in the third stage of gestation for isolation and cell culture. The umbilical cord of cutias assumes a spiral arrangement, with veins and arteries on it starting 50 days after coitus. The arteries present an outer layer of smooth muscle fibres in a longitudinal and circular arrangement and a medium layer of smooth muscle fibres with only longitudinal and intimate orientation and coated by the endothelium. The veins consist of longitudinal smooth muscle fibres with an extract of smooth muscle cells, and the endothelium, in all analysed gestational phases, is a structure bounded by simple pavement epithelial tissue originating from the amnion, adhered to Wharton’s Jelly and forming the umbilical vessels and allantoid duct. The proposed protocol allowed the collection of a high cellular concentration of umbilical cord progenitor cells from viable cutias.

A cutia vem sendo utilizada como modelo experimental em diversos estudos voltados à biologia reprodutiva. O cordão umbilical, anexo embrionário que une o feto à placenta, tem sido relatado como um importante sítio anatômico para obtenção de células-tronco. O objetivo deste estudo foi descrever macro e microscopicamente o cordão umbilical de cutias, em fases diferentes da gestação, expandir e cultivar in vitro as células progenitoras e relatar suas características morfológicas. Foram utilizadas sete cutias submetidas à cesariana para a coleta dos cordões umbilicais, cinco foram destinadas aos estudos da estrutura do cordão, em diferentes estágios de gestação (30, 35, 50, 75 e 100 dias pós-coito), e duas, no terço final da gestação, para isolamento e cultivo celular. O cordão umbilical de cutia assume disposição espiralada, com veias e artérias sobre ele a partir dos 50 dias após o coito. As artérias apresentam camada externa de fibras musculares lisas, disposição longitudinal e circular, camada média de fibras musculares lisas, apenas com disposição longitudinal e íntima revestida pelo endotélio. As veias constituídas por fibras musculares lisas longitudinais com um extrato de células musculares lisas e pelo endotélio. Em todas as fases gestacionais analisadas é uma estrutura delimitada por tecido epitelial simples pavimentoso, proveniente do âmnio, aderido a Geleia de Wharton e com formação de vasos umbilicais e ducto alantóide. O protocolo proposto permitiu a coleta de células progenitoras do cordão umbilical de cutias, viáveis com elevada concentração celular.