Rodrigo Panepucci | Universidade de São Paulo (original) (raw)
Papers by Rodrigo Panepucci
Frontiers in Oncology
Glioblastoma (GBM) is the most lethal and frequent type of brain tumor, leading patients to death... more Glioblastoma (GBM) is the most lethal and frequent type of brain tumor, leading patients to death in approximately 14 months after diagnosis. GBM treatment consists in surgical removal followed by radio and chemotherapy. However, tumors commonly relapse and the treatment promotes only a slight increase in patient survival. Thus, uncovering the cellular mechanisms involved in GBM resistance is of utmost interest, and the use of cell lines has been shown to be an extremely important tool. In this work, the exploration of RNAseq data from different GBM cell lines revealed different expression signatures, distinctly correlated with the behavior of GBM cell lines regarding proliferation indexes and radio-resistance. U87MG and U138MG cells, which presented expressively reduced proliferation and increased radio-resistance, showed a particular expression signature encompassing enrichment in many extracellular matrix (ECM) and receptor genes. Contrasting, U251MG and T98G cells, that presente...
Blood
4777 Introduction. Recently, several researchers have showed that mesenchymal stromal cells (MSCs... more 4777 Introduction. Recently, several researchers have showed that mesenchymal stromal cells (MSCs) are useful for preventing and treating the graft versus host disease, however the molecular mechanisms involved in immunomodulation of T lymphocytes by MSCs remains to be explored. Aims. With that in mind, we performed a whole genome microarray study to explore transcriptional differences in activated TCD4 and TCD8 lymphocytes immunomodulated or not by MSCs. Materials and Methods. Peripheral blood CD3+ T lymphocytes from 3 individuals were activated by anti-CD3/CD28 beads and cultured either in the absence or in the presence of MSC (5 to 1 ratio). Following a 5 day period, CD4+ and CD8+ lymphocytes cultivated or not with MSCs were immunomagnetically purified to over 95% purity (flow cytometry) and profiled by whole genome microarrays. Differentially expressed genes were analyzed in search of regulatory networks and levels of selected transcripts were evaluated by RT-PCR. Results. T lym...
Stem Cell Research & Therapy
Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs o... more Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. Methods: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. Results: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. Conclusions: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.
Background: Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies h... more Background: Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs carcinogenesis. In fact, several microRNAs have been described as aberrantly expressed in CRC tissues and in the serum of patients. More specifically, microRNAs with dual roles in both cancer and stem cell survival represent a potential source of novel molecular targets in CRC due to their described functions in normal and deregulated proliferation. However, the functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs involved in the control of pluripotency of stem cells in the proliferation and cell death of a colorectal cancer cell line. Methods: We performed transfection of 31 microRNA mimics in HCT116 CRC cells. Cell proliferation and cell death were measured after 4 days of treatment using fluorescence staining in a high content screening p...
Stem Cell Research & Therapy
Thoroughly understanding the molecular mechanisms responsible for the biological properties of pl... more Thoroughly understanding the molecular mechanisms responsible for the biological properties of pluripotent stem cells, as well as for the processes involved in reprograming, differentiation, and transition between Naïve and Primed pluripotent states, is of great interest in basic and applied research. Although pluripotent cells have been extensively characterized in terms of their transcriptome and miRNome, a comprehensive understanding of how these gene products specifically impact their biology, depends on gain-or loss-of-function experimental approaches capable to systematically interrogate their function. We review all studies carried up to date that used arrayed screening approaches to explore the function of these genetic elements on those biological contexts, using focused or genome-wide genetic libraries. We further discuss the limitations and advantages of approaches based on assays with population-level primary readouts, derived from single-parameter plate readers, or cell-level primary readouts, obtained using multiparametric flow cytometry or quantitative fluorescence microscopy (i.e., high-content screening). Finally, we discuss technical limitation and future perspectives, highlighting how the integration of screening data may lead to major advances in the field of stem cell research and therapy.
Blood Cells, Molecules, and Diseases
Scientific reports, Jan 15, 2017
Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-β can inhib... more Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-β can inhibit the polarization of naïve T cells into inflammatory Th17 cells, favoring the generation of stable iTregs, however the regulatory mechanisms involved are not fully understood. In this context, the roles of individual microRNAs in Tregs are largely unexplored. Naïve T cells were immunomagnetically isolated from umbilical cord blood and activated with anti-human CD2/CD3/CD28 beads in the presence of IL-2 alone (CD4Med) or with the addition of TGF-β and atRA (CD4TGF/atRA). As compared to CD4Med, the CD4TGF/atRA condition allowed the generation of highly suppressive CD4(+)CD25(hi)CD127(-)FOXP3(hi) iTregs. Microarray profiling allowed the identification of a set of microRNAs that are exclusively expressed upon TGF-β/atRA treatment and that are predicted to target a set of transcripts concordantly downregulated. This set of predicted targets were enriched for central components of IL-6/JAK/S...
American journal of physical medicine & rehabilitation, Jan 21, 2017
way, the following sentence “Moreover, there would be the added benefit of reduction in muscle da... more way, the following sentence “Moreover, there would be the added benefit of reduction in muscle damage and atrophy and less inflammation and pain...” should be reviewed. In addition, the effects of training on muscle performance, which are generally the most important effects among high-level athletes, could be achieved by simple exercise variations with similar training volume. For example, dynamic strength training has been shown to improve lower limb one-RM by approximately 60% (with three previous familiarization sessions in the one-RM test), which is slightly higher than reported by the authors for the lightemitting diode therapy after strength training (~53 and 37%, without familiarization sessions in the one-RM test). A greater magnitude of interleukin 1 beta gene expression was observed as inflammatory responses after 12 weeks of strength training and light-emitting diode therapy. Interestingly, inflammatory response and muscle damage are known to dramatically reduce after a few weeks of strength training, and Ferraresi et al. displayed similar results (decreasing creatine kinase and muscle soreness). The observed results can be explained by the short period (24 hrs) between the last training session and final muscle biopsy, due to a likely residual effect. In this way, do gene expression results represent another acute effect of training in the trained individuals rather than a chronic strength training adaptation? The authors did not address this rationale regarding evidence to assure that there is no negative interference from the interval of the muscle biopsy performed 24 hrs after the last training session. The authors also stated “On the other hand, patients suffering from inflammation, pain, loss of muscle mass and strength, and muscle atrophy as result of orthopaedic surgical procedures...”. Such an affirmation is an extrapolation of the study findings to a clinical setting that was not investigated, because the study comprises a strength training routine and not rehabilitation of injured individuals.
European journal of medical genetics, Jan 7, 2017
Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differ... more Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells we...
Stem cell research & therapy, Dec 30, 2016
Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces import... more Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces important challenges. For instance, increasing MSC migratory capacity as well as potentializing immune response suppression are of interest. For GvHD management, preventing opportunistic infections is also a valuable strategy, since immunocompromised patients are easy targets for infections. LL-37 is a host defense peptide (HDP) that has been deeply investigated due to its immunomodulatory function. In this scenario, the combination of MSC and LL-37 may result in a robust combination to be clinically used. In the present study, the effects of LL-37 upon the proliferation and migratory capacity of human placenta-derived MSCs (pMSCs) were assessed by MTT and wound scratch assays. The influence of LL-37 over the immunosuppressive function of pMSCs was then investigated using CFSE cell division kit. Flow cytometry and real-time PCR were used to investigate the molecular mechanisms involved in the ...
Molecular syndromology, 2017
Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving terminal chromosome 11q.... more Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving terminal chromosome 11q. The haploinsufficiency of multiple genes contributes to the overall clinical phenotype, which can include the variant Paris-Trousseau syndrome, a transient thrombocytopenia related to FLI1 hemizygous deletion. We investigated a boy with features of JBS using classic cytogenetic methods, FISH and high-resolution array CGH. The proband was found to have a mosaic ring chromosome 11 resulting in a hemizygous 11q terminal deletion of 8.6 Mb, leading to a copy number loss of 52 genes. The patient had a hemizygous deletion in the FLI1 gene region without apparent thrombocytopenia, and he developed diabetes mellitus type I, which has not previously been described in the spectrum of disorders associated with JBS. The relationship of some of the genes within the context of the phenotype caused by a partial deletion of 11q has provided insights concerning the developmental anomalies presented in t...
Blood, Jan 3, 2016
We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to i... more We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to immune response on outcomes after unrelated CB transplants (CBT). Pre-transplant DNA samples from 696 CBU with malignant diseases were genotyped for NLRP1, NLRP2, NLRP3, TIRAP/Mal, IL10, REL, TNFRSF1B and CTLA4. HLA compatibility was 6/6 in 10%, 5/6 in 39%, and ≥4/6 in 51% of transplants. Myeloablative conditioning was used in 80% and in vivo T-cell depletion in 81% of cases. The median number of total nucleated cells infused was 3.4x10(7)/kg. In multivariable analysis, Patients receiving CBU with GG-CTLA4 genotype had a poorer neutrophil recovery (HR: 1.33; p=0.02), increased non-relapse mortality (HR: 1.50; p<0.01) and inferior disease-free survival (HR: 1.41; p=0.02). We performed the same analysis in a more homogeneous subset of cohort 1 (cohort 2, n=305) of patients transplanted for acute leukemia, all given a myeloablative conditioning regimen and with available allele HLA typing...
BBA Clinical, 2016
Background: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells an... more Background: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. Methods: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. Results: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. Conclusions: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. General significance: MiRNAs are up-or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.
Experimental Cell Research, 2016
Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cell... more Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.
Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, Jan 22, 2016
Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype ... more Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quanti...
In Vitro Cellular & Developmental Biology - Animal, 2016
During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cell... more During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34(+) HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3β inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.
American Journal of Physical Medicine & Rehabilitation, 2016
V: Effects of light-emitting diode therapy on muscle hypertrophy, gene expression, performance, d... more V: Effects of light-emitting diode therapy on muscle hypertrophy, gene expression, performance, damage, and delayed-onset muscle soreness: case-control study with a pair of identical twins. Objective-The aim of this study was to verify how a pair of monozygotic twins would respond to light-emitting diode therapy (LEDT) or placebo combined with a strength-training program during 12 weeks. Design-This case-control study enrolled a pair of male monozygotic twins, allocated randomly to LEDT or placebo therapies. Light-emitting diode therapy or placebo was applied from a flexible light-emitting diode array (λ = 850 nm, total energy = 75 J, t = 15 seconds) to both quadriceps femoris muscles of each twin immediately after each strength training session (3 times/wk for 12
Blood Cells, Molecules, and Diseases, 2016
Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primar... more Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.
Frontiers in Oncology
Glioblastoma (GBM) is the most lethal and frequent type of brain tumor, leading patients to death... more Glioblastoma (GBM) is the most lethal and frequent type of brain tumor, leading patients to death in approximately 14 months after diagnosis. GBM treatment consists in surgical removal followed by radio and chemotherapy. However, tumors commonly relapse and the treatment promotes only a slight increase in patient survival. Thus, uncovering the cellular mechanisms involved in GBM resistance is of utmost interest, and the use of cell lines has been shown to be an extremely important tool. In this work, the exploration of RNAseq data from different GBM cell lines revealed different expression signatures, distinctly correlated with the behavior of GBM cell lines regarding proliferation indexes and radio-resistance. U87MG and U138MG cells, which presented expressively reduced proliferation and increased radio-resistance, showed a particular expression signature encompassing enrichment in many extracellular matrix (ECM) and receptor genes. Contrasting, U251MG and T98G cells, that presente...
Blood
4777 Introduction. Recently, several researchers have showed that mesenchymal stromal cells (MSCs... more 4777 Introduction. Recently, several researchers have showed that mesenchymal stromal cells (MSCs) are useful for preventing and treating the graft versus host disease, however the molecular mechanisms involved in immunomodulation of T lymphocytes by MSCs remains to be explored. Aims. With that in mind, we performed a whole genome microarray study to explore transcriptional differences in activated TCD4 and TCD8 lymphocytes immunomodulated or not by MSCs. Materials and Methods. Peripheral blood CD3+ T lymphocytes from 3 individuals were activated by anti-CD3/CD28 beads and cultured either in the absence or in the presence of MSC (5 to 1 ratio). Following a 5 day period, CD4+ and CD8+ lymphocytes cultivated or not with MSCs were immunomagnetically purified to over 95% purity (flow cytometry) and profiled by whole genome microarrays. Differentially expressed genes were analyzed in search of regulatory networks and levels of selected transcripts were evaluated by RT-PCR. Results. T lym...
Stem Cell Research & Therapy
Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs o... more Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. Methods: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. Results: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. Conclusions: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.
Background: Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies h... more Background: Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs carcinogenesis. In fact, several microRNAs have been described as aberrantly expressed in CRC tissues and in the serum of patients. More specifically, microRNAs with dual roles in both cancer and stem cell survival represent a potential source of novel molecular targets in CRC due to their described functions in normal and deregulated proliferation. However, the functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs involved in the control of pluripotency of stem cells in the proliferation and cell death of a colorectal cancer cell line. Methods: We performed transfection of 31 microRNA mimics in HCT116 CRC cells. Cell proliferation and cell death were measured after 4 days of treatment using fluorescence staining in a high content screening p...
Stem Cell Research & Therapy
Thoroughly understanding the molecular mechanisms responsible for the biological properties of pl... more Thoroughly understanding the molecular mechanisms responsible for the biological properties of pluripotent stem cells, as well as for the processes involved in reprograming, differentiation, and transition between Naïve and Primed pluripotent states, is of great interest in basic and applied research. Although pluripotent cells have been extensively characterized in terms of their transcriptome and miRNome, a comprehensive understanding of how these gene products specifically impact their biology, depends on gain-or loss-of-function experimental approaches capable to systematically interrogate their function. We review all studies carried up to date that used arrayed screening approaches to explore the function of these genetic elements on those biological contexts, using focused or genome-wide genetic libraries. We further discuss the limitations and advantages of approaches based on assays with population-level primary readouts, derived from single-parameter plate readers, or cell-level primary readouts, obtained using multiparametric flow cytometry or quantitative fluorescence microscopy (i.e., high-content screening). Finally, we discuss technical limitation and future perspectives, highlighting how the integration of screening data may lead to major advances in the field of stem cell research and therapy.
Blood Cells, Molecules, and Diseases
Scientific reports, Jan 15, 2017
Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-β can inhib... more Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-β can inhibit the polarization of naïve T cells into inflammatory Th17 cells, favoring the generation of stable iTregs, however the regulatory mechanisms involved are not fully understood. In this context, the roles of individual microRNAs in Tregs are largely unexplored. Naïve T cells were immunomagnetically isolated from umbilical cord blood and activated with anti-human CD2/CD3/CD28 beads in the presence of IL-2 alone (CD4Med) or with the addition of TGF-β and atRA (CD4TGF/atRA). As compared to CD4Med, the CD4TGF/atRA condition allowed the generation of highly suppressive CD4(+)CD25(hi)CD127(-)FOXP3(hi) iTregs. Microarray profiling allowed the identification of a set of microRNAs that are exclusively expressed upon TGF-β/atRA treatment and that are predicted to target a set of transcripts concordantly downregulated. This set of predicted targets were enriched for central components of IL-6/JAK/S...
American journal of physical medicine & rehabilitation, Jan 21, 2017
way, the following sentence “Moreover, there would be the added benefit of reduction in muscle da... more way, the following sentence “Moreover, there would be the added benefit of reduction in muscle damage and atrophy and less inflammation and pain...” should be reviewed. In addition, the effects of training on muscle performance, which are generally the most important effects among high-level athletes, could be achieved by simple exercise variations with similar training volume. For example, dynamic strength training has been shown to improve lower limb one-RM by approximately 60% (with three previous familiarization sessions in the one-RM test), which is slightly higher than reported by the authors for the lightemitting diode therapy after strength training (~53 and 37%, without familiarization sessions in the one-RM test). A greater magnitude of interleukin 1 beta gene expression was observed as inflammatory responses after 12 weeks of strength training and light-emitting diode therapy. Interestingly, inflammatory response and muscle damage are known to dramatically reduce after a few weeks of strength training, and Ferraresi et al. displayed similar results (decreasing creatine kinase and muscle soreness). The observed results can be explained by the short period (24 hrs) between the last training session and final muscle biopsy, due to a likely residual effect. In this way, do gene expression results represent another acute effect of training in the trained individuals rather than a chronic strength training adaptation? The authors did not address this rationale regarding evidence to assure that there is no negative interference from the interval of the muscle biopsy performed 24 hrs after the last training session. The authors also stated “On the other hand, patients suffering from inflammation, pain, loss of muscle mass and strength, and muscle atrophy as result of orthopaedic surgical procedures...”. Such an affirmation is an extrapolation of the study findings to a clinical setting that was not investigated, because the study comprises a strength training routine and not rehabilitation of injured individuals.
European journal of medical genetics, Jan 7, 2017
Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differ... more Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells we...
Stem cell research & therapy, Dec 30, 2016
Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces import... more Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces important challenges. For instance, increasing MSC migratory capacity as well as potentializing immune response suppression are of interest. For GvHD management, preventing opportunistic infections is also a valuable strategy, since immunocompromised patients are easy targets for infections. LL-37 is a host defense peptide (HDP) that has been deeply investigated due to its immunomodulatory function. In this scenario, the combination of MSC and LL-37 may result in a robust combination to be clinically used. In the present study, the effects of LL-37 upon the proliferation and migratory capacity of human placenta-derived MSCs (pMSCs) were assessed by MTT and wound scratch assays. The influence of LL-37 over the immunosuppressive function of pMSCs was then investigated using CFSE cell division kit. Flow cytometry and real-time PCR were used to investigate the molecular mechanisms involved in the ...
Molecular syndromology, 2017
Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving terminal chromosome 11q.... more Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving terminal chromosome 11q. The haploinsufficiency of multiple genes contributes to the overall clinical phenotype, which can include the variant Paris-Trousseau syndrome, a transient thrombocytopenia related to FLI1 hemizygous deletion. We investigated a boy with features of JBS using classic cytogenetic methods, FISH and high-resolution array CGH. The proband was found to have a mosaic ring chromosome 11 resulting in a hemizygous 11q terminal deletion of 8.6 Mb, leading to a copy number loss of 52 genes. The patient had a hemizygous deletion in the FLI1 gene region without apparent thrombocytopenia, and he developed diabetes mellitus type I, which has not previously been described in the spectrum of disorders associated with JBS. The relationship of some of the genes within the context of the phenotype caused by a partial deletion of 11q has provided insights concerning the developmental anomalies presented in t...
Blood, Jan 3, 2016
We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to i... more We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to immune response on outcomes after unrelated CB transplants (CBT). Pre-transplant DNA samples from 696 CBU with malignant diseases were genotyped for NLRP1, NLRP2, NLRP3, TIRAP/Mal, IL10, REL, TNFRSF1B and CTLA4. HLA compatibility was 6/6 in 10%, 5/6 in 39%, and ≥4/6 in 51% of transplants. Myeloablative conditioning was used in 80% and in vivo T-cell depletion in 81% of cases. The median number of total nucleated cells infused was 3.4x10(7)/kg. In multivariable analysis, Patients receiving CBU with GG-CTLA4 genotype had a poorer neutrophil recovery (HR: 1.33; p=0.02), increased non-relapse mortality (HR: 1.50; p<0.01) and inferior disease-free survival (HR: 1.41; p=0.02). We performed the same analysis in a more homogeneous subset of cohort 1 (cohort 2, n=305) of patients transplanted for acute leukemia, all given a myeloablative conditioning regimen and with available allele HLA typing...
BBA Clinical, 2016
Background: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells an... more Background: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. Methods: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. Results: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. Conclusions: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. General significance: MiRNAs are up-or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.
Experimental Cell Research, 2016
Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cell... more Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.
Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, Jan 22, 2016
Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype ... more Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quanti...
In Vitro Cellular & Developmental Biology - Animal, 2016
During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cell... more During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34(+) HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3β inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.
American Journal of Physical Medicine & Rehabilitation, 2016
V: Effects of light-emitting diode therapy on muscle hypertrophy, gene expression, performance, d... more V: Effects of light-emitting diode therapy on muscle hypertrophy, gene expression, performance, damage, and delayed-onset muscle soreness: case-control study with a pair of identical twins. Objective-The aim of this study was to verify how a pair of monozygotic twins would respond to light-emitting diode therapy (LEDT) or placebo combined with a strength-training program during 12 weeks. Design-This case-control study enrolled a pair of male monozygotic twins, allocated randomly to LEDT or placebo therapies. Light-emitting diode therapy or placebo was applied from a flexible light-emitting diode array (λ = 850 nm, total energy = 75 J, t = 15 seconds) to both quadriceps femoris muscles of each twin immediately after each strength training session (3 times/wk for 12
Blood Cells, Molecules, and Diseases, 2016
Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primar... more Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.