Sandra I. Anjo

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.

Short GeLC‐SWATH: A fast and reliable quantitative approach for proteomic screenings

The quantification of large proteomes across multiple samples has become the major focus of proteomics. In addition to the advantages of in‐gel digestion, the extensive time and sample handling required have precluded the use of this type of method for large quantitative screens. Therefore, an adaptation of the in‐gel digestion method, termed short‐GeLC, is proposed as a faster and more reproducible sample preparation method for quantitative approaches. The proposed methodology was compared with two well‐established procedures for sample preparation, GeLC‐MS and the classic liquid digestion followed by LC‐MS, using a membrane protein‐enriched sample. The results show that the short‐GeLC approach substantially reduces the amount of sample handling and the overall time required for analysis compared with the gel‐based methods without compromising the overall results at the protein identification level. Furthermore, the short‐GeLC approach in combination with the SWATH acquisition method leads to the best quantitative results: more proteins were quantified, and the reproducibility was improved. Finally, this method performed well even on challenging samples enriched in membrane proteins.

SWATH-MS as a tool for biomarker discovery: From basic research to clinical applications

In the era of quantitative proteomics, where mass spectrometry plays a pivotal role, in particular associated with the use of data‐independent acquisition, it is time to perform an overview of this growing field with special focus on one of the most promising approaches: SWATH‐MS, and to present future perspectives for its application as a translational tool. Therefore, a summary of this technique is presented focusing on two key relevant concepts associated with its application in biomarker discovery: the protein library and the individual digital maps concepts. It is also the purpose of this review to document the likely impact of SWATH‐MS in both fundamental and translational research including biomarker identification and creation of diagnostic tools. To that end, the two concepts referred above were integrated with ongoing technical developments. Finally, some of the current restrictions for the implementation of SWATH‐MS on a large scale are identified, and potential solutions presented, namely protocol standardization combined with the use of the proper standards.

Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson’s Disease

Research in the last decade strongly suggests that mesenchymal stem cell (MSC)‐mediated therapeutic benefits are mainly due to their secretome, which has been proposed as a possible therapeutic tool for the treatment of Parkinsons disease (PD). Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail (i.e., conditioned medium). In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons (i.e., tyrosine hydroxylase‐positive cells) and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome (through combined mass spectrometry analysis and Bioplex assays) revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. Stem Cells Translational Medicine 2017;6:634–646

Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation

In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.

Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome?

IntroductionThe use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear.MethodsIn the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells.ResultsThe present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome.ConclusionsOur results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

Mesenchymal Stem Cell Secretome: A Potential Tool for the Prevention of Muscle Degenerative Changes Associated With Chronic Rotator Cuff Tears:

Background: Massive rotator cuff tears (MRCTs) are usually chronic lesions with pronounced degenerative changes, where advanced fatty degeneration and atrophy can make the tear irreparable. Human mesenchymal stem cells (hMSCs) secrete a range of growth factors and vesicular systems, known as secretome, that mediates regenerative processes in tissues undergoing degeneration. Purpose: To study the effect of hMSC secretome on muscular degenerative changes and shoulder function on a rat MRCT model. Study Design: Controlled laboratory study. Methods: A bilateral 2-tendon (supraspinatus and infraspinatus) section was performed to create an MRCT in a rat model. Forty-four Wistar-Han rats were randomly assigned to 6 groups: control group (sham surgery), lesion control group (MRCT), and 4 treated-lesion groups according to the site and periodicity of hMSC secretome injection: single local injection, multiple local injections, single systemic injection, and multiple systemic injections. Forelimb function was analyzed with the staircase test. Atrophy and fatty degeneration of the muscle were evaluated at 8 and 16 weeks after injury. A proteomic analysis was conducted to identify the molecules present in the hMSC secretome that can be associated with muscular degeneration prevention. Results: When untreated for 8 weeks, the MRCT rats exhibited a significantly higher fat content (0.73% ± 0.19%) compared with rats treated with a single local injection (0.21% ± 0.04%; P < .01) or multiple systemic injections (0.25% ± 0.10%; P < .05) of hMSC secretome. At 16 weeks after injury, a protective effect of the secretome in the multiple systemic injections (0.62% ± 0.14%; P < .001), single local injection (0.76% ± 0.17%; P < .001), and multiple local injections (1.35% ± 0.21%; P < .05) was observed when compared with the untreated MRCT group (2.51% ± 0.42%). Regarding muscle atrophy, 8 weeks after injury, only the single local injection group (0.0993% ± 0.0036%) presented a significantly higher muscle mass than that of the untreated MRCT group (0.0794% ± 0.0047%; P < .05). Finally, the proteomic analysis revealed the presence of important proteins with muscle regeneration, namely, pigment epithelium-derived factor and follistatin. Conclusion: The study data suggest that hMSC secretome effectively decreases the fatty degeneration and atrophy of the rotator cuff muscles. Clinical Relevance: We describe a new approach for decreasing the characteristic muscle degeneration associated with chronic rotator cuff tears. This strategy is particularly important for patients whose tendon healing after later surgical repair could be compromised by the progressing degenerative changes. In addition, both precise intramuscular local injection and multiple systemic secretome injections have been shown to be promising delivery forms for preventing muscle degeneration.

Unraveling Mesenchymal Stem Cells’ Dynamic Secretome Through Nontargeted Proteomics Profiling

The modulatory and regenerative potential shown by the use of MSC secretomes has emphasized the importance of their proteomics profiling. Proteomic analysis, initially focused on the targeted analysis of some candidate proteins or the identification of the secreted proteins, has been changing to an untargeted profiling also based on the quantitative evaluation of the secreted proteins.The study of the secretome can be accomplished through several different proteomics-based approaches; however this analysis must overcome one key challenge of secretome analysis: the low amount of secreted proteins and usually their high dilution.In this chapter, a general workflow for the untargeted proteomic profile of MSCs secretome is presented, in combination with a comprehensive description of the major techniques/procedures that can be used. Special focus is given to the main procedures to obtain the secreted proteins, from secretome concentration by ultrafiltration to protein precipitation. Lastly, different proteomics-based approaches are presented, emphasizing alternative digestion techniques and available mass spectrometry-based quantitative methods.