The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold's intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp), a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases.

Histamine is commonly acknowledged as an inflammatory mediator in peripheral tissues, leaving its role in brain immune responses scarcely studied. Therefore, our aim was to uncover the cellular and molecular mechanisms elicited by this molecule and its receptors in microglia-induced inflammation by evaluating cell migration and inflammatory mediator release.

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

Retinoids regulate a wide spectrum of cellular functions from the embryo throughout adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits make retinoids very attractive molecules for medical purposes. In light of some of the physicochemical limitations of retinoids, the development of drug delivery systems offers several advantages for clinical translation of retinoid-based therapies, including improved solubilization, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In this Review, we discuss advances in preclinical and clinical tests regarding retinoid formulations, specifically the ones based in natural retinoids, evaluated in the context of regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations of retinoid formulations, as well as prospects to push the field forward, will be presented.

Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently-labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent-based compound (saponin) or cholesterol-modification of the miRNA, Exo-Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT-PCR data showed that, compared with native sEVs, Exo-Fect modulation led to a > 1000-fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR-155-5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo-Fect-loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo-Fect-loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial-glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types.

As mediators of intercellular communication, extracellular vesicles containing molecular cargo, such as microRNAs, are secreted by cells and taken up by recipient cells to influence their cellular phenotype and function. Here we report that cardiac stress-induced differential microRNA content, with miR-200c-3p being one of the most enriched, in cardiomyocyte-derived extracellular vesicles mediates functional cross-talk with endothelial cells. Silencing of miR-200c-3p in mice subjected to chronic increased cardiac pressure overload resulted in attenuated hypertrophy, smaller fibrotic areas, higher capillary density, and preserved cardiac ejection fraction. We were able to maximally rescue microvascular and cardiac function with very low doses of antagomir, which specifically silences miR-200c-3p expression in non-myocyte cells. Our results reveal vesicle transfer of miR-200c-3p from cardiomyocytes to cardiac endothelial cells, underlining the importance of cardiac intercellular communication in the pathophysiology of heart failure.

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

C-terminal binding proteins (CtBP) are transcriptional co-repressors regulating gene expression. CtBP promote neuronal survival through repression of pro-apoptotic genes, and may represent relevant targets for neurodegenerative disorders, such as Parkinson's disease (PD). Nevertheless, evidence of the role of CtBP1 and CtBP2 in neurodegeneration are scarce. Herein, we showed that CtBP1 and CtBP2 are expressed in neurons, dopaminergic neurons, astrocytes, and microglia in the substantia nigra (SN) and striatum of adult mice. Old mice showed a lower expression of CtBP1 in the SN and higher expression of CtPB2 in the SN and striatum compared with adult mice. In vivo models for PD (paraquat, MPTP, 6-OHDA) showed increased expression of CtBP1 in the SN and striatum while CtBP2 expression was increased in the striatum of paraquat-treated rats only. Moreover, an increased expression of both CtBP was found in a dopaminergic cell line (N27) exposed to 6-OHDA. In the 6-OHDA PD model, we found a dual effect using an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB): higher concentrations (e.g. 2500 µM, 1000 µM) inhibited dopaminergic survival, while at 250 μM it counteracted cell death. In vitro, this latter protective role was absent after the siRNA silencing of CtBP1 or CtBP2. Altogether, this is the first report exploring the cellular and regional expression pattern of CtBP in the nigrostriatal pathway and the neuroprotective role in PD toxin-based models. CtBP could counteract dopaminergic cell death in the 6-OHDA PD model and, therefore, CtBP function and therapeutic potential in PD should be further explored.

Externally triggered drug delivery systems hold considerable promise for improving the treatment of many diseases, in particular, diseases where the spatial-temporal release of the drug is critical to maximize their biological effect whilst minimizing undesirable, off-target, side effects.

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

Parkinson's disease is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra with no effective cure available. MicroRNA-124 has been regarded as a promising therapeutic entity for Parkinson's disease due to its pro-neurogenic and neuroprotective roles. However, its efficient delivery to the brain remains challenging. Here, we used umbilical cord blood mononuclear cell-derived extracellular vesicles as a biological vehicle to deliver microRNA (miR)-124-3p and evaluate its therapeutic effects in a mouse model of Parkinson's disease. In vitro, miR-124-3p-loaded small extracellular vesicles induced neuronal differentiation in subventricular zone neural stem cell cultures and protected N27 dopaminergic cells against 6-hydroxydopamine-induced toxicity. In vivo, intracerebroventricularly administered small extracellular vesicles were detected in the subventricular zone lining the lateral ventricles and in the striatum and substantia nigra, the brain regions most affected by the disease. Most importantly, although miR-124-3p-loaded small extracellular vesicles did not increase the number of new neurons in the 6-hydroxydopamine-lesioned striatum, the formulation protected dopaminergic neurons in the substantia nigra and striatal fibers, which fully counteracted motor behavior symptoms. Our findings reveal a novel promising therapeutic application of small extracellular vesicles as delivery agents for miR-124-3p in the context of Parkinson's disease.

More effective approaches are needed in the treatment of blood cancers, in particular acute myeloid leukemia (AML), that are able to eliminate resistant leukemia stem cells (LSCs) at the bone marrow (BM), after a chemotherapy session, and then enhance hematopoietic stem cell (HSC) engraftment for the re-establishment of the HSC compartment. Here, we investigate whether light-activatable nanoparticles (NPs) encapsulating all-trans-retinoic acid (RA+NPs) could solve both problems. Our in vitro results show that mouse AML cells transfected with RA+NPs differentiate towards antitumoral M1 macrophages through RIG.1 and OASL gene expression. Our in vivo results further show that mouse AML cells transfected with RA+NPs home at the BM after transplantation in an AML mouse model. The photo-disassembly of the NPs within the grafted cells by a blue laser enables their differentiation towards a macrophage lineage. This macrophage activation seems to have systemic anti-leukemic effect within the BM, with a significant reduction of leukemic cells in all BM compartments, of animals treated with RA+NPs, when compared with animals treated with empty NPs. In a separate group of experiments, we show for the first time that normal HSCs transfected with RA+NPs show superior engraftment at the BM niche than cells without treatment or treated with empty NPs. This is the first time that the activity of RA is tested in terms of long-term hematopoietic reconstitution after transplant using an in situ activation approach without any exogenous priming or genetic conditioning of the transplanted cells. Overall, the approach documented here has the potential to improve consolidation therapy in AML since it allows a dual intervention in the BM niche: to tackle resistant leukemia and improve HSC engraftment at the same time.

Methamphetamine (METH) is a psychostimulant drug of abuse that causes severe brain damage. However, the mechanisms responsible for these effects are poorly understood, particularly regarding the impact of METH on hippocampal neurogenesis. Moreover, neuropeptide Y (NPY) is known to be neuroprotective under several pathological conditions. Here, we investigated the effect of METH on dentate gyrus (DG) neurogenesis, regarding cell death, proliferation and differentiation, as well as the role of NPY by itself and against METH-induced toxicity. DG-derived neurosphere cultures were used to evaluate the effect of METH or NPY on cell death, proliferation or neuronal differentiation. Moreover, the role of NPY and its receptors (Y(1), Y(2) and Y(5)) was investigated under conditions of METH-induced DG cell death. METH-induced cell death by both apoptosis and necrosis at concentrations above 10 nM, without affecting cell proliferation. Furthermore, at a non-toxic concentration (1 nM), METH decreased neuronal differentiation. NPY's protective effect was mainly due to the reduction of glutamate release, and it also increased DG cell proliferation and neuronal differentiation via Y(1) receptors. In addition, while the activation of Y(1) or Y(2) receptors was able to prevent METH-induced cell death, the Y(1) subtype alone was responsible for blocking the decrease in neuronal differentiation induced by the drug. Taken together, METH negatively affects DG cell viability and neurogenesis, and NPY is revealed to be a promising protective tool against the deleterious effects of METH on hippocampal neurogenesis.

Leukaemia cells that are resistant to conventional therapies are thought to reside in protective niches. Here, we describe light-inducible polymeric retinoic acid (RA)-containing nanoparticles (NPs) with the capacity to accumulate in the cytoplasm of leukaemia cells for several days and release their RA payloads within a few minutes upon exposure to blue/UV light. Compared to NPs that are not activated by light exposure, these NPs more efficiently reduce the clonogenicity of bone marrow cancer cells from patients with acute myeloid leukaemia (AML) and induce the differentiation of RA-low sensitive leukaemia cells. Importantly, we show that leukaemia cells transfected with light-inducible NPs containing RA can engraft into bone marrow in vivo in the proximity of other leukaemic cells, differentiate upon exposure to blue light and release paracrine factors that modulate nearby cells. The NPs described here offer a promising strategy for controlling distant cell populations and remotely modulating leukaemic niches.

There is a high quest for novel therapeutic strategies to enhance recovery after stroke. MicroRNA-124 (miR-124) has been described as neuroprotective and anti-inflammatory molecule. Moreover, miR-124 is a well described enhancer of adult neurogenesis that could offer potentially beneficial effects. Herein, we used miR-124-loaded nanoparticles (miR-124 NPs) to evaluate their therapeutic potential in an in vitro and in vivo model of stroke. For that, neuroprotective and neurogenic responses were assessed in an in vitro model of stroke. Here, we found that miR-124 NPs decreased cell death and improved neuronal differentiation of subventricular zone (SVZ) neural stem cell cultures after oxygen and glucose deprivation. In contrast, intravenous injection of miR-124 NPs immediately after permanent focal ischemia induced by photothrombosis (PT) did not provide a better neurological outcome. In addition, treatment did not affect the number of 5-bromo-2'-deoxyuridine (BrdU)- and doublecortin/BrdU- positive cells in the SVZ at the study endpoint of 14 days after PT. Likewise, the ischemic insult did not affect the numbers of neuronal progenitors in the SVZ. However, in PT mice miR-124 NPs were able to specifically augment interleukin-6 levels at day 2 post-stroke. Furthermore, we also showed that NPs reached the brain parenchyma and were internalized by brain resident cells. Although, promising in vitro data could not be verified in vivo as miR-124 NPs treatment did not improve functional outcome nor presented beneficial actions on neurogenesis or post-stroke inflammation, we showed that our NP formulation can be a safe alternative for drug delivery into the brain.

Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP) as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2). RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state), promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases.