Functionalized Hydrogels for Cartilage Repair: The Value of Secretome-Instructive Signaling.

Cartilage repair has been a challenge in the medical field for many years. Although treatments that alleviate pain and injury are available, none can effectively regenerate the cartilage. Currently, regenerative medicine and tissue engineering are among the developed strategies to treat cartilage injury. The use of stem cells, associated or not with scaffolds, has shown potential in cartilage regeneration. However, it is currently known that the effect of stem cells occurs mainly through the secretion of paracrine factors that act on local cells. In this review, we will address the use of the secretome-a set of bioactive factors (soluble factors and extracellular vesicles) secreted by the cells-of mesenchymal stem cells as a treatment for cartilage regeneration. We will also discuss methodologies for priming the secretome to enhance the chondroregenerative potential. In addition, considering the difficulty of delivering therapies to the injured cartilage site, we will address works that use hydrogels functionalized with growth factors and secretome components. We aim to show that secretome-functionalized hydrogels can be an exciting approach to cell-free cartilage repair therapy.

Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells.

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Alternative Methods as Tools for Obesity Research: In Vitro and In Silico Approaches.

The study of adipogenesis is essential for understanding and treating obesity, a multifactorial problem related to body fat accumulation that leads to several life-threatening diseases, becoming one of the most critical public health problems worldwide. In this review, we propose to provide the highlights of the adipogenesis study based on in vitro differentiation of human mesenchymal stem cells (hMSCs). We list in silico methods, such as molecular docking for identification of molecular targets, and in vitro approaches, from 2D, more straightforward and applied for screening large libraries of substances, to more representative physiological models, such as 3D and bioprinting models. We also describe the development of physiological models based on microfluidic systems applied to investigate adipogenesis in vitro. We intend to identify the main alternative models for adipogenesis evaluation, contributing to the direction of preclinical research in obesity. Future directions indicate the association of in silico and in vitro techniques to bring a clear picture of alternative methods based on adipogenesis as a tool for obesity research.

Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation.

Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.

Targeting L-Proline Uptake as New Strategy for Anti-chagas Drug Development.

L-Proline is an important amino acid for the pathogenic protists belonging to Trypanosoma and Leishmania genera. In Trypanosoma cruzi, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses. In this study, we explore the L-Proline uptake as a chemotherapeutic target for T. cruzi. Novel inhibitors have been proposed containing the amino acid with a linker and a variable region able to block the transporter. A series of sixteen 1,2,3-triazolyl-proline derivatives have been prepared for in vitro screening against T. cruzi epimastigotes and proline uptake assays. We successfully obtained inhibitors that interfere with the amino acid internalization, which validated our design targeting the metabolite's transport. The presented structures are one of few examples of amino acid transporter inhibitors. The unprecedent application of this strategy on the development of new chemotherapy against Chagas disease, opens a new horizon on antiparasitic drug development against parasitic diseases and other pathologies.

Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells.

BACKGROUND: Human adipose-derived stromal/stem cells (hASCs) are one of the most useful types of mesenchymal stromal/stem cells, which are adult multipotent cells with great therapeutic potential for the treatment of several diseases. However, for successful clinical application, it is critical that high-quality cells can be obtained. Diverse factors seem to be able to influence cell quality and performance, especially factors related to donors' intrinsic characteristics, such as age. Nevertheless, there is no consensus regarding this characteristic, and there is conflicting information in the literature. AIM: To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors. METHODS: hASCs were harvested from liposuctioned adipose tissue obtained from female donors (aged 20-70 years). Cells were distributed into two groups according to age range: old hASCs (oASCs, ≥ 55 years, n = 9) and young hASCs (yASCs, ≤ 35 years, n = 9). For each group, immunophenotypic characterization was performed by flow cytometry. Population doubling time was assessed over seven days. For adipogenic potential evaluation, lipid deposits were assessed after 7 d, 14 d and 21 d of adipogenic induction. Osteogenic potential was verified by analyzing cell mineralization after 14 d, 21 d and 28 d of osteogenic induction. mRNA expression of PPARγ2, CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction. RESULTS: hASCs were successfully obtained, cultured, and grouped according to their age: yASCs (26.33 ± 4.66 years old) and oASCs (64.78 ± 4.58 years old). After maintenance of the cells in culture, there were no differences in morphology between cells from the young and old donors. Additionally, both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells. The average doubling time indicated that yASCs (4.09 ± 0.94 d) did not significantly differ from oASCs (4.19 ± 1.29 d). Concerning differentiation potential, after adipogenic and osteogenic induction, yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells. However, no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis. Additionally, the mRNA expression of PPARγ2, CEBPA and Runx2 were similar in yASCs and oASCs. CONCLUSION: Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.

Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation.

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.

L-Glutamine uptake is developmentally regulated and is involved in metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas disease, can obtain L-glutamine (Gln) through the enzyme glutamine synthetase (GS) using glutamate (Glu) and ammonia as substrates. In this work, we show additional non-canonical roles for this amino acid: its involvement in ATP maintenance and parasite survival under severe metabolic stress conditions and its participation in the differentiation process occurring in the insect vector (metacyclogenesis). These roles are dependent on the supply of Gln from an extracellular source. We show that T. cruzi incorporates Gln through a saturable and specific transport system, which results in unusual stability at elevated temperatures. The activity was moderately higher at pH values between 6 and 7 and was sensitive to the dissipation of the H+ gradient at the plasma membrane. When analysed in the different life cycle stages, we found that Gln transport is developmentally regulated. In fact, Gln uptake and GS activity seem to be finely regulated at most stages: when GS activity is increased, transport is decreased and vice versa, with the exception of trypomastigotes, where both sources of Gln are diminished. This metabolic adaptation reflects the relevance of Gln in T. cruzi biology and the plasticity of these parasites to adjust their metabolism to changing environments.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection.

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/ mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/ mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle.

The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake.

Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite's replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE) with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport), which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi.

Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed) remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC50 172.6 µM). Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction) and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca(2+) and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC50 31 µM). Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote) were more affected as were the processes of differentiation and cell invasion.

The uniqueness of the Trypanosoma cruzi mitochondrion: opportunities to identify new drug target for the treatment of Chagas disease.

Trypanosoma cruzi is the causative agent of Chagas' disease, which affects some 8 - 10 million people in the Americas. The only two drugs approved for the etiological treatment of the disease in humans were launched more than 40 years ago and have serious drawbacks. In the present work, we revisit the unique characteristics of T. cruzi mitochondria and mitochondrial metabolism. The possibility of taking advantage of these peculiarities to target new drugs against this parasite is also discussed.