Alessandra Melo de Aguiar

Dissimilar Differentiation of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood, and Adipose Tissue:

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 ± 943 and 243.89 ± 145.52 μm2 per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 μm2 for BM-derived MSCs and 2.36 μm2 for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.

Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

Are purified or expanded cord blood-derived CD133+ cells better at improving cardiac function?

Endothelial progenitor cells (EPCs), which express the CD133 marker, can differentiate into mature endothelial cells (ECs) and create new blood vessels. Normal angiogenesis is unable to repair the injured tissues that result from myocardial infarction (MI). Patients who have high cardiovascular risks have fewer EPCs and their EPCs exhibit greater in vitro senescence. Human umbilical cord blood (HUCB)-derived EPCs could be an alternative to rescue impaired stem cell function in the sick and elderly. The aim of this study was to purify HUCB-derived CD133+ cells, expand them in vitro and evaluate the efficacy of the purified and expanded cells in treating MI in rats. CD133+ cells were selected for using CD133-coupled magnetic microbeads. Purified cells stained positive for EPC markers. The cells were expanded and differentiated in media supplemented with fetal calf serum and basic fibroblast growth factor, insulin-like growth factor-I and vascular endothelial growth factor (VEGF). Differentiation was confirmed by lack of staining for EPC markers. These expanded cells exhibited increased expression of mature EC markers and formed tubule-like structures in vitro. Only the expanded cells expressed VEGF mRNA. Cells were expanded up to 70-fold during 60 days of culture, and they retained their functional activity. Finally, we evaluated the therapeutic potential of purified and expanded CD133+ cells in treating MI by intramyocardially injecting them into a rat model of MI. Rats were divided into three groups: A (purified CD133+ cells-injected); B (expanded CD133+ cells-injected) and C (saline buffer-injected). We observed a significant improvement in left ventricular ejection fraction for groups A and B. In summary, CD133+ cells can be purified from HUCB, expanded in vitro without loosing their biological activity, and both purified and expanded cells show promising results for use in cellular cardiomyoplasty. However, further pre-clinical testing should be performed to determine whether expanded CD133+ cells have any clinical advantages over purified CD133+ cells.

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.

PUMILIO-2 Is Involved in the Positive Regulation of Cellular Proliferation in Human Adipose-Derived Stem Cells

Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes.

The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test

Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models. For RC rat-only weight regression, both cells had the same accordance (50%), while for RC rat-only millimole regression, the accordance was 50% for ADSC and 42% for 3T3s. Thus, ADSC have similar capability for GHS class prediction as the 3T3 cell line for the evaluated reference substances. Therefore, ADSCs showed the potential to be considered a novel model for use in evaluating cytotoxicity in drug development and industry as well as for regulatory purposes to reduce or replace the use of laboratory animals with acceptable sensitivity for toxicity prediction in humans. These cells can be used to complete the results from other models, mainly because of its human origin. Moreover, it is less expensive in comparison with other existing models.

Polysome Profiling Shows the Identity of Human Adipose-Derived Stromal/Stem Cells in Detail and Clearly Distinguishes Them from Dermal Fibroblasts

Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that ∼ 1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies.

Secretome from resident cardiac stromal cells stimulates proliferation, cardiomyogenesis and angiogenesis of progenitor cells

In the heart, tissue-derived signals play a central role on recruiting/activating stem cell sources to induce cardiac lineage specification for maintenance of tissue homeostasis and repair. Cardiac resident stromal cells (CRSCs) may play a pivotal role in cardiac repair throughout their secretome. Here, we performed the characterization of CRSCs and their secretome by analyzing the composition of their culture-derived extracellular matrix (ECM) and conditioned medium (CM) and by investigating their potential effect on adipose-derived stem cell (ADSC) and progenitor cell behavior. We confirmed that CRSCs are a heterogeneous cell population whose secretome is composed by proteins related to cellular growth, immune response and cardiovascular development and function. We also observed that CRSC secretome was unable to change the behavior of ADSCs, except for proliferation. Additionally, CM from CRSCs demonstrated the potential to drive proliferation and cardiac differentiation of H9c2 cells and also the ability to induce angiogenesis in vitro. Our data suggest that the CRSCs can be a source of important modulating signals for cardiac progenitor cell recruitment/activation.