Signal transduction pathways alter the molecular cargo of extracellular vesicles: implications in regenerative medicine.

Extracellular vesicles (EVs) possess regenerative properties and are also considered as future vaccines. All types of cells secrete EVs; however, the amount of EVs secreted by the cells varies under various physiological as well as pathological states. Several articles have reviewed the molecular composition and potential therapeutic applications of EVs. Likewise, the 'sorting signals' associated with specific macromolecules have also been identified, but how the signal transduction pathways prevailing in the parent cells alter the molecular profile of the EVs or the payload they carry has not been sufficiently reviewed. Here, we have specifically discussed the implications of these alterations in the macromolecular cargo of EVs for their therapeutic applications in regenerative medicine.

Beyond animal models: revolutionizing neurodegenerative disease modeling using 3D in vitro organoids, microfluidic chips, and bioprinting.

Neurodegenerative diseases (NDs) are characterized by uncontrolled loss of neuronal cells leading to a progressive deterioration of brain functions. The transition rate of numerous neuroprotective drugs against Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, leading to FDA approval, is only 8-14% in the last two decades. Thus, in spite of encouraging preclinical results, these drugs have failed in human clinical trials, demonstrating that traditional cell cultures and animal models cannot accurately replicate human pathophysiology. Hence, in vitro three-dimensional (3D) models have been developed to bridge the gap between human and animal studies. Such technological advancements in 3D culture systems, such as human-induced pluripotent stem cell (iPSC)-derived cells/organoids, organ-on-a-chip technique, and 3D bioprinting, have aided our understanding of the pathophysiology and underlying mechanisms of human NDs. Despite these recent advances, we still lack a 3D model that recapitulates all the key aspects of NDs, thus making it difficult to study the ND's etiology in-depth. Hence in this review, we propose developing a combinatorial approach that allows the integration of patient-derived iPSCs/organoids with 3D bioprinting and organ-on-a-chip technique as it would encompass the neuronal cells along with their niche. Such a 3D combinatorial approach would characterize pathological processes thoroughly, making them better suited for high-throughput drug screening and developing effective novel therapeutics targeting NDs.

Priming mesenchymal stromal cells with neurotrophic factors boosts the neuro-regenerative potential of their secretome.

Aim: To explore the neuroprotective potential of the secretome (conditioned medium, CM) derived from neurotrophic factors-primed mesenchymal stromal cells (MSCs; primed CM) using an endoplasmic reticulum (ER) stress-induced in vitro model system. Methods: Establishment of ER-stressed in vitro model, immunofluorescence microscopy, real-time PCR, western blot. Results: Exposure of ER-stressed Neuro-2a cells to the primed-CM significantly restored the neurite outgrowth parameters and improved the expression of neuronal markers like Tubb3 and Map2a in them compared with the naive CM. Primed CM also suppressed the induction of apoptotic markers Bax and Sirt1, inflammatory markers Cox2 and NF-κB, and stress kinases such as p38 and SAPK/JNK in the stress-induced cells. Conclusion: The secretome from primed MSCs significantly restored ER stress-induced loss of neuro-regenesis.

Endoplasmic reticulum (ER) stress-mediated accumulation of misfolded protein is one of the causes involved in the onset of several neurodegenerative diseases (ND). Under physiological conditions, ER stress activates the unfolded protein response (UPR) that repairs the misfolded proteins. However, if the ER stress becomes chronic, the UPR fails to repair the misfolded proteins leading to disease conditions such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, etc. Most in vitro systems are based on the infliction of acute ER stress on the target cells, which kills them, and thus, are not physiologically relevant models, as their neuro-regeneration is not possible. Here, we have developed a physiologically relevant in vitro model system, wherein we exposed Neuro-2a cells to an ER stress inducer which significantly affected their neuro-regenesis without killing them. These dysfunctional cells were then used to assess the effect of secretome (conditioned medium, CM) derived from mesenchymal stromal cells (MSCs) primed or not with neurotrophic factors. We found that priming of MSCs with neurotrophic factors enhances their neuroprotective potential. We demonstrate that when primed CM is given either as a single dose or in multiple doses, it restores neuro-regenesis and protects the stressed Neuro-2a cells from cell death. More importantly, the restoration of neuro-regenesis by primed CM is significantly higher as compared with the naive CM. These results clearly underscore the importance of priming the MSCs with neurotrophic factors for developing more effective therapeutic strategies to combat ND.

Secretome of Young Mesenchymal Stromal Cells Rejuvenates Aged Mesenchymal Stromal Cells by Normalizing Their Phenotype and Restoring Their Differentiation Profile.

During aging, the proliferation and differentiation ability of mesenchymal stem/stromal cells (MSCs) gets affected, and hence, aged MSCs are not preferred for regenerative purposes. Rapid identification of aging-associated changes within MSCs and the mechanistic pathways involved are necessary to determine optimal cell sources to treat musculoskeletal disorders in older patients. In the present study, we have identified a set of phenotypic markers, namely downregulated expression of CD90 and upregulated expression of CD45, as age-defining markers for the bone marrow-derived MSCs. We also show that these phenotypic changes in aged MSCs correlate with their aging-mediated differentiation defects. We find that oxidative stress signaling leading to the activation of nuclear factor kappa B (NF-κB) plays an essential role in altering the phenotype and differentiation ability of the aged MSCs. We further show that treatment of aged MSCs with the conditioned medium (CM) derived from young MSCs (young-CM) restored their phenotype and differentiation potential to the young-like by ameliorating activation of NF-κB signaling in them. Similar changes could also be achieved by using an inhibitor of NF-κB signaling, showing that oxidative stress-induced NF-κB activation is the causative factor in the aging of MSCs. Additionally, we show that treating young MSCs with hydrogen peroxide mimics all the aging-mediated changes in them, underscoring the involvement of oxidative stress in the aging of MSCs. Overall, our data suggest that the altered expression of CD90 and CD45 surface markers can be used as a primary screen to identify the onset of aging in the MSCs, which can be quickly reversed by their in vitro treatment with young-CM or NF-κB inhibitor. Our study also puts the phenotypic characterization of MSCs in a clinical perspective.

Mesenchymal stromal cells-derived secretome protects Neuro-2a cells from oxidative stress-induced loss of neurogenesis.

Neurodegenerative diseases (ND) are characterized by debilitating medical conditions that principally affect the neuronal cells in the human brain. One of the major reasons that there are no effective drugs for the treatment of ND is because researchers face technical challenges while conducting studies to understand the molecular mechanism behind ND. Although various studies have established in vitro neurodegenerative model systems, we feel that these model systems are not physiologically relevant, as they do not mimic the in vivo situation of chronic insult. Therefore, the primary aim of this study was to establish an in vitro neurodegenerative model system by inducing oxidative stress in such a way that the neuronal cells remain viable, but lose their structural and functional characteristics. Using a murine neuroblastoma cell line, Neuro-2a, we demonstrate that induction of oxidative stress significantly affects various neurite outgrowth parameters and reduces the expression of neuronal and autophagy markers without causing apoptosis in them. Previously, we have discussed the possible therapeutic applications of mesenchymal stromal cells (MSCs) and their secretome in the treatment of ND. Here, using two distinct approaches, we show that when Neuro-2a cells subjected to oxidative stress are exposed to MSC-derived conditioned medium (secretome), they exhibit a significant improvement in various neuronal parameters and in the expression of neuronal markers. Overall, our findings support the salutary role of MSC-derived secretome in rescuing the oxidative stress-induced loss of neurogenesis using a physiologically relevant in vitro model system. Our data underscore the propensity of the MSC-secretome in reversing ND.

Extracellular vesicles isolated from mesenchymal stromal cells primed with neurotrophic factors and signaling modifiers as potential therapeutics for neurodegenerative diseases.

Neurodegenerative diseases are characterized by a progressive and irreversible loss of neuronal cells leading to cognitive impairments and memory loss. Despite being a powerful tool for clinical applications, the use of mesenchymal stromal cells (MSCs) imposes several challenges in terms of delivery, safety and variability. MSCs exert their regenerative effects through a paracrine mode of action, also known as the secretome that is composed of cytokines, chemokines, growth factors, proteins and extracellular vesicles - namely the microvesicles and the exosomes. It has been reported that preconditioning of MSCs alters the molecular composition of their secretome, thereby improving their therapeutic potential. Based on our previous work and other reports, we propose a unique strategy, comprising of the following parameters, for harnessing the true potential of the extracellular vesicles isolated from the primed MSCs for promoting neuroregeneration: i) examining the signaling mechanisms prevailing in the MSCs, ii) assessing the age of the MSC donor, iii) priming MSCs with neurotrophic factors and examining the expression of neuronal and autophagy markers in them, and iv) examining the extracellular vesicles for autophagy-promoting-neurotrophic factors. We speculate that our strategy may provide an impetus for improving the efficacy of MSCs in reversing the process of neurodegeneration.