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 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) 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.

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.

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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.

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.

The adipogenic process is characterized by the expression of adipocyte differentiation markers that lead to changes in cell metabolism and to the accumulation of lipid droplets. Moreover, during early adipogenesis, cells undergo a strong downregulation of translational activity with a decrease in cell size, proliferation and migration. In the present study, we identified that after 24 hours of adipogenic induction, human adipose tissue-derived stem cells (hASCs) undergo a G1-cell cycle arrest consistent with reduced proliferation, and this effect was correlated with a shift in polysome profile with an enrichment of the monosomal fraction and a reduction of the polysomal fraction. Polysome profiling analysis also revealed that this change in the monosomal/polysomal ratio was related to a strong downregulation of cell cycle and proliferation genes, such as cyclins and cyclin-dependent kinases (CDKs). Comparing total and polysome-associated mRNA sequencing, we also observed that this downregulation was mostly due to a reduction of cell cycle and proliferation transcripts via control of total mRNA abundance, rather than by translational control.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs.

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.