Stem cell extracellular vesicles for neural regeneration

In the last decade, multipotent mesenchymal stromal cells (MSCs) demonstrated a significant therapeutic efficacy, particularly in cell therapy approaches aiming at tissue regeneration. MSCs exert their action via trophic support, induction of angiogenesis, immunomodulation and reduction of necrosis at affected tissues. Importantly, these regenerative and protective properties are largely associated to MSC secretome. Unfortunately, cell-based approaches not always meet the criteria for a smooth translation to the clinic. For instance, the use of stem cells in pathologies with a very short therapeutic window, such as few hours, is not compatible with the requested minimal criteria for MSC release, before administration to the patient. Notwithstanding, in the regenerative medicine field, the MSC mechanism of action paradigm was recently extended to include the action of extracellular vesicles (EVs), which are cytoplasm-containing cellular bodies secreted by a wide range of cell types. Intriguingly, many studies reported that EVs generated by MSCs are able to recapitulate the majority of the regenerative properties of parental MSCs. Starting from these premises, the objectives of the present doctoral research project were: to address EV-mediated cell-to-cell communication as novel MSC mechanism of action; to address reprogrammed MSC-EV generation; to define, for the first time in the literature, stem cell EV molecular content (e.g.: miRNome), comparing reprogrammed to non-reprogrammed MSC-EVs; to challenge stem cell-EV therapeutic potential in a model of acute tissue damage, as a proof-of-concept for feasibility and effectiveness of a stem cell-based albeit cell-free regenerative strategy. Intriguingly, EVs may be produced in a ready-to-use formulation, so that clinicians could use them as soon as a therapeutic need arises, also in the case of an urgent one. In this way, EV-shuttled MSC regenerative properties could exert beneficial effects also on pathologies currently lacking any cell therapy option. To develop this innovative therapeutic strategy, MSCs were isolated from different tissues and their biological properties were evaluated in order to choose the MSC source most suitable for the implementation of the project. Thus, both MSC transcriptome and immunophenotype were addressed. MSCs from adult sources (e.g.: bone marrow) showed senescence-related features in vitro, correlated to donor’s age in vivo. On the other hand, MSCs from perinatal tissues (e.g.: cord blood) showed a phenotype more similar to that of pericytes, which are the in vivo progenitors of MSCs. Therefore, cord blood was chosen as MSC source, also in the prospective of clinical translation, since public banking of cord blood units for clinical use already exists worldwide. Next, thanks to an extended analysis of the stromal populations present in cord blood, a MSC subpopulation showing higher proliferation properties and significantly longer telomeres was isolated. In addition, the standard cord blood MSC isolation protocol was improved, leading to an efficiency of 80%. Eventually, MSC secretome-associated anti-inflammatory and anti-apoptotic properties were observed in vitro and in vivo. In order to investigate if EVs contributed to MSC paracrine properties, MSC-EV secretion and regenerative properties were assessed. The MSC-EV therapeutic effectiveness was challenged in an in vitro model of acute tissue damage. Intriguingly, MSC-EVs could rescue damage-induced cell mortality, showing the same protective effect of parental MSCs. In spite of the use of a high proliferative cord blood MSC subpopulation, primary cultures still show a limited lifespan. In order to increase their replicative potential and to better exploit their EV production, induced cellular reprogramming was tested on MSCs as an alternative to traditional immortalization techniques. In this way, MSC-derived cell lines endowed with unlimited lifespan were generated, and permanent modification of their genome was avoided. The next step was to confirm the generation of EVs from reprogrammed MSCs, since reprogramming drastically changes cell identity. Furthermore, the EV miRNome load of reprogrammed and non-reprogrammed MSCs was addressed. Importantly, the majority of miRNAs were common between the two samples, indicating that reprogramming did not change the EV miRNA content. This result could have relevant consequences on the functional features of reprogrammed MSC-EVs, since EV-mediated miRNA transfer from donor to target cells was proposed as one of MSC mechanisms of action. In the last part of this doctoral research, stem cell (non-reprogrammed and reprogrammed MSCs)-EV therapeutic effectiveness was addressed and compared to that of parental MSCs. In order to do that, an organotypic ex vivo mouse model of brain ischemia was used. This model recapitulated the modulation of some ischemic damage-related parameters, including increased secretion of inflammatory cytokines, high tissue necrosis and the impairment of neuronal and astrocytic cell populations. Therefore, this model mimicked early phase events of brain ischemia, whose thrombolytic clinical treatment must be administered within 3-6 hours of first signs of ischemia. Notably, stem cell-EVs were tested for the first time in this pathological context to verify their potential role in tissue regeneration. Strikingly, stem cell-EV administration to affected tissues showed significant neuroprotective properties, which were comparable to those of parental MSCs. Importantly, the ischemic damage-related parameters previously described were rescued. In particular, inflammatory-associated parameters underwent the most statistically significant decrease, showing levels similar to or better than those of the uninjured brain tissue. This is of uttermost importance, considering that chronic inflammation is detrimental to tissue regeneration. To conclude, the results of the present PhD thesis confirmed the feasibility of stem cell EV-based therapies in regenerative medicine approaches. In the future, this innovative EV therapy may be applied to pathological contexts currently without a cell therapy option. In the framework of advanced therapy medicinal products, the new drug would be the EVs, rather than the parental stem cells. Finally, EVs could play the role of ready-to-use anti-inflammatory molecule carriers, in order to guarantee a rapid therapeutic action for the regeneration of injured tissues

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail

Age-related changes in the energy of human mesenchymal stem cells

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells

Mitochondrial dysfunction in Parkinsonian mesenchymal stem cells impairs differentiation

Sporadic cases account for 90-95% of all patients with Parkinson's Disease (PD). Atypical Parkinsonism comprises approximately 20% of all patients with parkinsonism. Progressive Supranuclear Palsy (PSP) belongs to the atypical parkinsonian diseases and is histopathologically classified as a tauopathy. Here, we report that mesenchymal stem cells (MSCs) derived from the bone marrow of patients with PSP exhibit mitochondrial dysfunction in the form of decreased membrane potential and inhibited NADH-dependent respiration. Furthermore, mitochondrial dysfunction in PSP-MSCs led to a significant increase in mitochondrial ROS generation and oxidative stress, which resulted in decrease of major cellular antioxidant GSH. Additionally, higher basal rate of mitochondrial degradation and lower levels of biogenesis were found in PSP-MSCs, together leading to a reduction in mitochondrial mass. This phenotype was biologically relevant to MSC stemness properties, as it heavily impaired their differentiation into adipocytes, which mostly rely on mitochondrial metabolism for their bioenergetic demand. The defect in adipogenic differentiation was detected as a significant impairment of intracellular lipid droplet formation in PSP-MSCs. This result was corroborated at the transcriptional level by a significant reduction of PPARγ and FABP4 expression, two key genes involved in the adipogenic molecular network. Our findings in PSP-MSCs provide new insights into the etiology of 'idiopathic' parkinsonism, and confirm that mitochondrial dysfunction is important to the development of parkinsonism, independent of the type of the cell

Extracellular vesicles as graft biomarkers to address lung transplantation outcome

Lung transplantation is the last therapeutic option for end-stage pulmonary failure. Yet, clinical complications may rise after transplantation, such as primary grafts dysfunction (PGD) or chronic lung allograft dysfunction (CLAD). Current clinical parameters have failed to assess the quality of the graft and to predict transplantation outcome. Extracorporeal photopheresis (ECP) is a treatment for graft-versus-host disease. Peripheral blood white blood cells (WBC) are isolated, exposed to 8-methoxypsoralen photosensitizing agent, and subsequently treated with ultraviolet radiation before reinfusion into the patient, causing massive WBC apoptosis. Our working hypothesis is that extracellular vesicles (EV) produced by either the pre-transplantation organ (donor) or host (recipient) could be non-invasive biomarkers to evaluate tissue damage at the cellular level and to monitor organ engraftment

Removing exogenous information using pedigree data

Management of certain populations requires the preservation of its pure genetic background. When, for different reasons, undesired alleles are introduced, the original genetic conformation must be recovered. The present study tested, through computer simulations, the power of recovery (the ability for removing the foreign information) from genealogical data. Simulated scenarios comprised different numbers of exogenous individuals taking partofthe founder population anddifferent numbers of unmanaged generations before the removal program started. Strategies were based on variables arising from classical pedigree analyses such as founders? contribution and partial coancestry. The ef?ciency of the different strategies was measured as the proportion of native genetic information remaining in the population. Consequences on the inbreeding and coancestry levels of the population were also evaluated. Minimisation of the exogenous founders? contributions was the most powerful method, removing the largest amount of genetic information in just one generation.However, as a side effect, it led to the highest values of inbreeding. Scenarios with a large amount of initial exogenous alleles (i.e. high percentage of non native founders), or many generations of mixing became very dif?cult to recover, pointing out the importance of being careful about introgression events in populatio

FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks

A circular RNA map for human induced pluripotent stem cells of foetal origin

Background Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. Methods Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. • View related content for this article Findings Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. Interpretation A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation

Are Farm-Reared Quails for Game Restocking Really Common Quails (Coturnix coturnix)?: A Genetic Approach

The common quail (Coturnix coturnix) is a popular game species for which restocking with farm-reared individuals is a common practice. In some areas, the number of released quails greatly surpasses the number of wild breeding common quail. However, common quail are difficult to raise in captivity and this casts suspicion about a possible hybrid origin of the farmed individuals from crosses with domestic Japanese quail (C. japonica). In this study we used a panel of autosomal microsatellite markers to characterize the genetic origin of quails reared for hunting purposes in game farms in Spain and of quails from an experimental game farm which was founded with hybrids that have been systematically backcrossed with wild common quails. The genotypes of these quail were compared to those of wild common quail and domestic strains of Japanese quail. Our results show that more than 85% of the game farm birds were not common quail but had domestic Japanese quail ancestry. In the experimental farm a larger proportion of individuals could not be clearly separated from pure common quails. We conclude that the majority of quail sold for restocking purposes were not common quail. Genetic monitoring of individuals raised for restocking is indispensable as the massive release of farm-reared hybrids could represent a severe threat for the long term survival of the native species

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field