A. Listoni - Academia.edu (original) (raw)
Papers by A. Listoni
By the pluripotency induction, adult somatic cells acquire very similar behavior to embryonic ste... more By the pluripotency induction, adult somatic cells acquire very similar behavior to embryonic stem cells, eliminating ethical issues related to the use of such research. However, the mechanisms involved in induced pluripotent stem (iPS) cells are not yet fully elucidated. The aim of this study was to induce pluripotency in rabbit adipose stem (ADS) cells. Rabbit ADS cells were collected and characterized by their morphology, dynamic growth, differentiation potential, viability under cryopreservation and phenotypic profile. The ADS cells were transduced with a lentiviral vector containing four human or murine pluripotency factors (OCT4, KLF4, SOX2 and c-MYC). Cells were maintained in IMDM medium, supplemented with 10% bovine fetal serum for six days, when they were trypsinized and 5x103 cells were platted on mitomycin-treated murine embryonic fibroblasts (MEFs) feeder layers with iPS medium. Several protocols were tested to acquire the pluripotent state, including the addition of MEK and GSK inhibitors or human LIF, but just partial reprogramming was detected. In order to verify the integration efficiency of pluripotent factors into the rabbit ADS cells, ADS cells were transduced with only one lentiviral vector containing an individual factor (OCT4 or SOX2), or with a combination of the two factors, those were conjugated with fluorescent reporters, vexGFP (OCT4) and mCitrine (SOX2). The integration efficiency was analyzed and sorted by flow cytometer, and analyzed by confocal microscopy. Rabbit ADS cells were viable after cryopreservation and demonstrated a mesenchymal stem cell phenotype as fibroblastic morphology, differentiation potential in adipocytes, osteocytes and chondrocytes and a positive expression of CD73 and CD90 and negative expression of CD34 and CD45. An exceptional high proliferation potential was observed. The pluripotency induction resulted in only partial reprogramming morphology in all protocols tested. By the integration efficiency assay, OCT4 factor was detected into the cells, but not the SOX2. Rabbit ADS are easy and fast to collect, isolate and proliferate in vitro and share the same characteristics of other mesenchymal stem cells, with a exceptional high proliferation rate. The pluripotent state could not be reached using several strategies, and just partial reprogramming cells were detected. By the integration assay, we verified that the OCT4 factor was properly transduced into the ADS cell, but the SOX2 factor could not be detected. The failure in the pluripotency induction and in the integration of SOX2 into the cells remains unclear, and further studies are necessary to better explain the mechanisms involved in the reprogramming. A possible link between proliferation and integration of SOX2 should be further investigated. We expect that managing the proliferation rate of rabbit ADS cells, the iPS cells can be generated. Sponsored by FAPESP.
Pesquisa Veterinária Brasileira, 2014
Prestes 2014. [Comparison of the biochemical composition of equine amniotic fluid collected in di... more Prestes 2014. [Comparison of the biochemical composition of equine amniotic fluid collected in different gestational phases and at delivery.] Comparação da composição bioquímica do líquido amniótico equino colhido em diferentes estágios gestacionais e no momento do parto. Pesquisa Veterinária Brasileira 34 :00-00. The viability and fetal maturity can be estimated by biochemical evaluation of the fetal fluids of several species; however the biochemical composition of amniotic fluid during pregnancy is not fully defined for equine. The aim of this study was to establish and compare the biochemical profile of amniotic fluid in different moments of pregnancy and at delivery, in order to better explain the peculiarities of the physiology of pregnancy in mares. The values founded for pH, osmolarity, glucose, urea, creatinine, gamma-GT, Sodium, potassium, chloride and total protein were evaluated in amniotic fluid collected from 122 mares comparing the results between the initial-third (IT), mid-third (MT) and latter--third (FT) of gestation and at delivery (D). The gestational period samples were collected at slaughterhouses. Gestational ages (IG), in days, were defined following the regression formula suggested by by measuring the craniocaudal (CC) distance of the fetuses. Commercial kits for biochemical evaluation were used. Due to the presence of varying degrees of asymmetry and deviations from a standard Gaussian distribution, the Kruskal-Wallis test was used to compare the median of each response variable among the study groups. When there has been significant evidence that at least one of the medians differed from the others, the Wilcoxon test was used to perform multiple comparisons between groups. The Bonferroni method was used to adjust the resulting p-value for multiple comparisons. Statistical analysis was performed using PROC Npar1way and statistical significance was defined as p<0.05. The pH and osmolarity of equine amniotic fluid did not change significantly during the stages of pregnancy and at delivery. The values found for glucose were significantly lower during late pregnancy and at delivery. Concentrations of urea tended to be statistically different in at least one of the groups. A significant increase in creatinine concentrations was observed during the initial-third, medium-third and the final-third of pregnancy and the value found at delivery remained equal to final-third. Values for Gamma GT differed only between FT and D groups and more studies should be conducted about its role in the amniotic fluid of domestic species. For the sodium and chloride 1 Recebido em 22 de setembro de 2013.
Stem Cell Discovery, 2014
ABSTRACT Several studies with mesenchymal stem cells (MSCs) have been developed in many species b... more ABSTRACT Several studies with mesenchymal stem cells (MSCs) have been developed in many species because of its ability to differentiate into other mesoderm lineages, capacity of self-regeneration, low immunogenicity, paracrine, anti-inflammatory, immunomodulatory and antiapoptotic effects which make them a promissory source to be used in therapeutic strategies. The aim of this study is to report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes and its processing and cultivation. For this, after anesthetic block from the region corresponding to the CT, bone marrow harvesting was performed with a myelogram’s needle. The samples collected showed plastic adherence with 96 hours and took approximately 32 days to reach 80% confluence. And then differentiation into adipogenic and osteogenic lineages was performed. Samples showed morphological changes during differentiation protocol, but not all presented production of extracellular deposits of calcium or intracellular fat droplets. The anatomical site tested showed to be an alternative site of harvest of BM once provided with the appropriate isolation and culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and with great security. Based on this, it can be concluded that CT is an excellent anatomical site for isolation and culture of MSCs and the proposed technique is viable and feasible to be held in buffaloes.
Stem Cell Research & Therapy, 2014
Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodula... more Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.
Cell Transplantation, 2010
Adipose tissue may represent a potential source of adult stem cells for tissue engineering applic... more Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.
Reproduction, Fertility and Development, 2013
ABSTRACT Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-depe... more ABSTRACT Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle's salts+10% FCS, FSH, and LH, in a 5% CO(2) atmosphere. To delay meiosis, the oocytes were maintained for 6h in medium in the presence of 25µM BL-I+6.25µM ROS. The oocytes were then cultured for 18h in agent-free medium to resume meiosis, completing 24h of maturation. After 24h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO(2) atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2×10(6)spermmL(-1). The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa+0.6% BSA+2.5% FCS in a 5% CO(2), 5% O(2), and 90% N(2) atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey's test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3±3.74%; BL-I+ROS, 38.0±4.5%. Thus, the presence of BL-I+ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.
By the pluripotency induction, adult somatic cells acquire very similar behavior to embryonic ste... more By the pluripotency induction, adult somatic cells acquire very similar behavior to embryonic stem cells, eliminating ethical issues related to the use of such research. However, the mechanisms involved in induced pluripotent stem (iPS) cells are not yet fully elucidated. The aim of this study was to induce pluripotency in rabbit adipose stem (ADS) cells. Rabbit ADS cells were collected and characterized by their morphology, dynamic growth, differentiation potential, viability under cryopreservation and phenotypic profile. The ADS cells were transduced with a lentiviral vector containing four human or murine pluripotency factors (OCT4, KLF4, SOX2 and c-MYC). Cells were maintained in IMDM medium, supplemented with 10% bovine fetal serum for six days, when they were trypsinized and 5x103 cells were platted on mitomycin-treated murine embryonic fibroblasts (MEFs) feeder layers with iPS medium. Several protocols were tested to acquire the pluripotent state, including the addition of MEK and GSK inhibitors or human LIF, but just partial reprogramming was detected. In order to verify the integration efficiency of pluripotent factors into the rabbit ADS cells, ADS cells were transduced with only one lentiviral vector containing an individual factor (OCT4 or SOX2), or with a combination of the two factors, those were conjugated with fluorescent reporters, vexGFP (OCT4) and mCitrine (SOX2). The integration efficiency was analyzed and sorted by flow cytometer, and analyzed by confocal microscopy. Rabbit ADS cells were viable after cryopreservation and demonstrated a mesenchymal stem cell phenotype as fibroblastic morphology, differentiation potential in adipocytes, osteocytes and chondrocytes and a positive expression of CD73 and CD90 and negative expression of CD34 and CD45. An exceptional high proliferation potential was observed. The pluripotency induction resulted in only partial reprogramming morphology in all protocols tested. By the integration efficiency assay, OCT4 factor was detected into the cells, but not the SOX2. Rabbit ADS are easy and fast to collect, isolate and proliferate in vitro and share the same characteristics of other mesenchymal stem cells, with a exceptional high proliferation rate. The pluripotent state could not be reached using several strategies, and just partial reprogramming cells were detected. By the integration assay, we verified that the OCT4 factor was properly transduced into the ADS cell, but the SOX2 factor could not be detected. The failure in the pluripotency induction and in the integration of SOX2 into the cells remains unclear, and further studies are necessary to better explain the mechanisms involved in the reprogramming. A possible link between proliferation and integration of SOX2 should be further investigated. We expect that managing the proliferation rate of rabbit ADS cells, the iPS cells can be generated. Sponsored by FAPESP.
Pesquisa Veterinária Brasileira, 2014
Prestes 2014. [Comparison of the biochemical composition of equine amniotic fluid collected in di... more Prestes 2014. [Comparison of the biochemical composition of equine amniotic fluid collected in different gestational phases and at delivery.] Comparação da composição bioquímica do líquido amniótico equino colhido em diferentes estágios gestacionais e no momento do parto. Pesquisa Veterinária Brasileira 34 :00-00. The viability and fetal maturity can be estimated by biochemical evaluation of the fetal fluids of several species; however the biochemical composition of amniotic fluid during pregnancy is not fully defined for equine. The aim of this study was to establish and compare the biochemical profile of amniotic fluid in different moments of pregnancy and at delivery, in order to better explain the peculiarities of the physiology of pregnancy in mares. The values founded for pH, osmolarity, glucose, urea, creatinine, gamma-GT, Sodium, potassium, chloride and total protein were evaluated in amniotic fluid collected from 122 mares comparing the results between the initial-third (IT), mid-third (MT) and latter--third (FT) of gestation and at delivery (D). The gestational period samples were collected at slaughterhouses. Gestational ages (IG), in days, were defined following the regression formula suggested by by measuring the craniocaudal (CC) distance of the fetuses. Commercial kits for biochemical evaluation were used. Due to the presence of varying degrees of asymmetry and deviations from a standard Gaussian distribution, the Kruskal-Wallis test was used to compare the median of each response variable among the study groups. When there has been significant evidence that at least one of the medians differed from the others, the Wilcoxon test was used to perform multiple comparisons between groups. The Bonferroni method was used to adjust the resulting p-value for multiple comparisons. Statistical analysis was performed using PROC Npar1way and statistical significance was defined as p<0.05. The pH and osmolarity of equine amniotic fluid did not change significantly during the stages of pregnancy and at delivery. The values found for glucose were significantly lower during late pregnancy and at delivery. Concentrations of urea tended to be statistically different in at least one of the groups. A significant increase in creatinine concentrations was observed during the initial-third, medium-third and the final-third of pregnancy and the value found at delivery remained equal to final-third. Values for Gamma GT differed only between FT and D groups and more studies should be conducted about its role in the amniotic fluid of domestic species. For the sodium and chloride 1 Recebido em 22 de setembro de 2013.
Stem Cell Discovery, 2014
ABSTRACT Several studies with mesenchymal stem cells (MSCs) have been developed in many species b... more ABSTRACT Several studies with mesenchymal stem cells (MSCs) have been developed in many species because of its ability to differentiate into other mesoderm lineages, capacity of self-regeneration, low immunogenicity, paracrine, anti-inflammatory, immunomodulatory and antiapoptotic effects which make them a promissory source to be used in therapeutic strategies. The aim of this study is to report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes and its processing and cultivation. For this, after anesthetic block from the region corresponding to the CT, bone marrow harvesting was performed with a myelogram’s needle. The samples collected showed plastic adherence with 96 hours and took approximately 32 days to reach 80% confluence. And then differentiation into adipogenic and osteogenic lineages was performed. Samples showed morphological changes during differentiation protocol, but not all presented production of extracellular deposits of calcium or intracellular fat droplets. The anatomical site tested showed to be an alternative site of harvest of BM once provided with the appropriate isolation and culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and with great security. Based on this, it can be concluded that CT is an excellent anatomical site for isolation and culture of MSCs and the proposed technique is viable and feasible to be held in buffaloes.
Stem Cell Research & Therapy, 2014
Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodula... more Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.
Cell Transplantation, 2010
Adipose tissue may represent a potential source of adult stem cells for tissue engineering applic... more Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.
Reproduction, Fertility and Development, 2013
ABSTRACT Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-depe... more ABSTRACT Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle's salts+10% FCS, FSH, and LH, in a 5% CO(2) atmosphere. To delay meiosis, the oocytes were maintained for 6h in medium in the presence of 25µM BL-I+6.25µM ROS. The oocytes were then cultured for 18h in agent-free medium to resume meiosis, completing 24h of maturation. After 24h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO(2) atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2×10(6)spermmL(-1). The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa+0.6% BSA+2.5% FCS in a 5% CO(2), 5% O(2), and 90% N(2) atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey's test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3±3.74%; BL-I+ROS, 38.0±4.5%. Thus, the presence of BL-I+ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.