Mesenchymal Stem Cells and Their Derivatives: Old Problems and New Possibilities in Regenerative Medicine for Neurological Diseases

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)-derived EVs (MSC-EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC-EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long-term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow-derived MSC lineages. MSC-EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC-EVs, postischemic immunosuppression (i.e., B-cell, natural killer cell, and T-cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC-EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof-of-concept studies in stroke patients. Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC-induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long-term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post-stroke immune responses. Also, because EVs are well-tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.

Mesenchymal stem cell (MSC) transplantation has been shown to represent a potential treatment for traumatic spinal cord injury (SCI). However, there are several obstacles that need to be overcome before MSCs can be considered for clinical application, such as failure of MSCs to reach the spinal cord lesion core and possible tumor formation. Recent studies have suggested that MSC treatment is beneficial owing to paracrine-secreted factors. Extracellular vesicles are considered to be some of the most valuable paracrine molecules. However, the therapeutic mechanism of extracellular vesicles on spinal cord injury has not been studied clearly. Therefore, our study investigated the effect of systemic administration of extracellular vesicles on the loss of motor function after SCI and examined the potential mechanisms underlying their effects. Disruption of the blood-spinal cord barrier (BSCB) is a crucial factor that can be detrimental to motor function recovery. Pericytes are an important component of the neurovascular unit, and play a pivotal role in maintaining the structural integrity of the BSCB. Our study demonstrated that administration of bone mesenchymal stem cell-derived extracellular vesicles (BMSC-EV) reduced brain cell death, enhanced neuronal survival and regeneration, and improved motor function compared with the administration of BMSC-EV free culture media (EV-free CM). Besides, the BSCB was attenuated and pericyte coverage was significantly decreased in vivo. Furthermore, we found that exosomes reduced pericyte migration via downregulation of NF-κB p65 signaling, with a consequent decrease in the permeability of the BSCB. In summary, we identified that extracellular vesicles treatment suppressed the migration of pericytes and further improved the integrity of the BSCB via NF-κB p65 signaling in pericytes. Our data suggest that extracellular vesicles may serve as a promising treatment strategy for SCI.

Objective: Mesenchymal stem cells (MSCs) are isolated from various human tissues and used for therapy, in which beneficial effects are attributed mainly to mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Whereas MSCs of diverse tissue types share cardinal stem cell features, it is becoming evident that MSCs of each tissue type possess unique properties as well. For designing efficient stem cellbased therapies, it is crucial to understand the unique properties associated with MSCs and MSC-EVs of each tissue type. Such unique properties can be analyzed through transcriptomic approaches using comprehensive gene expression databases and sophisticated analytical tools. Here, we comparatively studied the transcriptomes in MSC-EVs of dental pulp and adipose tissue. Additionally, the transcriptomes of MSC-EVs were compared with the cellular transcriptomes of MSCs for the same tissue types. Methods: MSCs were cultured from human dental pulp and adipose tissue specimens. Conditioned culture media were collected to prepare MSC-EVs, from which RNAs were isolated and subjected to next-generation sequencing for transcriptomic analysis. Gene expression signatures in MSC-EVs of each tissue type were investigated using gene set analysis. Results: MSC-EVs obtained from dental pulp-derived MSCs showed distinct transcriptomic signatures of neurogenesis and neural retina development while MSC-EVs of adipose tissue-derived MSCs showed signatures of mitochondrial activity and skeletal system development. The transcriptomes of MSC-EVs resembled the cellular transcriptomes of MSCs, and the genes associated with neurogenesis were highly expressed in both MSCs and MSC-EVs of dental pulp. Adipose tissue-derived MSCs and MSC-EVs highly expressed genes associated with angiogenesis, hair growth, and dermal matrices. Conclusion: The clear and distinct signatures of neurogenesis and neural retina development in dental pulp-derived MSC-EVs imply neurodegenerative disorders and retinal diseases as putative therapeutic targets. In contrast, the transcripts in adipose tissue-derived MSC-EVs could be useful in rejuvenating the skin and musculoskeletal system. Further insights into MSC-EVs of divergent tissue types may expand the list of potential therapeutic targets.

What's in a Name?

Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicle Conditioning Has an Immunosuppressive Effect on NK Cells

Ultraviolet B (UVB) radiation is a major cause of aging in dermal fibroblasts. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show antioxidant activity. In this study, the anti-aging effects of MSC-EVs on dermal fibroblast photoaging induced by UVB radiation were evaluated, and the effects of extracellular vesicles derived from dermal fibroblasts (Fb-EVs) were compared. Human umbilical cord mesenchymal stem cells and human dermal fibroblasts were cultured, and MSC-EVs and Fb-EVs were isolated and characterized. Human dermal fibroblasts were cultured in the absence or presence of different concentrations of EVs 24 hours prior to UVB radiation exposure. Cell proliferation and cell cycle were evaluated, and senescent cells and intracellular ROS were detected. The expressions of matrix metalloproteinase-1 (MMP-1), extracellular matrix protein collagen type 1 (Col-1), and antioxidant proteins such as glutathione peroxidase 1 (GPX-1), superoxide dismutase (SOD), and catalase were also analyzed. Pretreatment with MSC-EVs or Fb-EVs significantly inhibited the production of ROS induced by UVB radiation, increased dermal fibroblast proliferation, protected cells against UVB-induced cell death and cell cycle arrest, and remarkably decreased the percentage of aged cells. Pretreatment with MSC-EVs or Fb-EVs promoted the expressions of GPX-1 and Col-1 and decreased the expression of MMP-1. Both MSC-EVs and Fb-EVs protected dermal fibroblasts from UVB-induced photoaging, likely through their antioxidant activity.

Background: Some studies have reported results from the use of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) to treat osteoarthritis (OA). Objective: To evaluate the efficacy of MSC-EVs as a treatment for OA. Data sources: Databases were searched using the terms 'mesenchymal stem cells', 'osteoarthritis' and 'extracellular vesicles.' Study eligibility criteria: Studies performed in animal models utilizing MSC-EVs to treat OA that described the macroscopic evaluation or histological evaluation were included. Study appraisal: The quality of the studies was examined using the CAMARADES quality checklist. Results: MSC-EVs were superior to the placebo in the macroscopic evaluation and histological evaluation. MSC-EVs were more effective in the early stage of OA and once a week was better than multiple times a week. Limitations: The included studies were highly heterogeneous. Conclusion: MSC-EVs may improve the results of macroscopic and histological evaluations of OA.

The goal of this study was to determine the role of microRNA transfer in mediating the effects of mesenchymal stem cell-derived extracellular vesicles in acute lung injury. Experimental cell and animal studies. University-based research laboratory. THP-1 monocytes, bone marrow-derived macrophages, and C57BL/6 mice. To determine the microRNA transfer in vitro, mesenchymal stem cells and mesenchymal stem cell-derived extracellular vesicles were cultured with THP-1 cells and bone marrow-derived macrophages and then assayed for microRNA expression in the target cells. To examine the role of microRNA transfer in vivo, mesenchymal stem cell-derived extracellular vesicles were administered to mice with lipopolysaccharide-induced lung injury. Mesenchymal stem cell-derived extracellular vesicles were efficiently taken up by macrophages in vitro and in vivo. miR-27a-3p was one of the most highly expressed microRNAs in THP-1 cells in microarray analysis and was transferred from mesenchymal stem cells and mesenchymal stem cell-derived extracellular vesicles to THP-1/bone marrow-derived macrophages. Mesenchymal stem cell-derived extracellular vesicles promoted M2 polarization in bone marrow-derived macrophages, which was inhibited by lentiviral anti-miR-27a-3p transduction. Mesenchymal stem cell-derived extracellular vesicles administered systemically and intratracheally were as effective as mesenchymal stem cells in alleviating acute lung injury, elevating miR-27a-3p levels in alveolar macrophages, and promoting M2 macrophage polarization. Treatment of mesenchymal stem cell-derived extracellular vesicles concurrently decreased alveolar macrophage expression of nuclear factor kappa B subunit 1, a target of miR-27a-3p. Lentiviral transduction of mesenchymal stem cells with anti-miR-27a-3p or knockdown of miR-27a-3p in vivo abolished the effects of mesenchymal stem cell-derived extracellular vesicles on acute lung injury and M2 macrophage polarization. Mesenchymal stem cell-derived extracellular vesicles mitigate acute lung injury at least partially via transferring miR-27a-3p to alveolar macrophages. miR-27a-3p acts to target NFKB1 and is a crucial regulator of M2 macrophage polarization.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

Mesenchymal cells were described for their potency to interact with human immune cells and to modulate thereby immune responses. Besides mesenchymal stromal cells (MSC) from diverse tissue sources, like bone marrow and umbilical cord, also mesenchymal adherent cells within the heart tissue were able to attenuate and modulate induced immune cell activation or inflammatory processes. Mechanistic studies with MSC support the hypothesis, that mainly paracrine acting molecules, in particular extracellular vesicles (EVs), are responsible for the observed functional effects. EVs are known as potent intercellular communicators by delivering proteins, lipids, RNA and other small signaling molecules to a recipient cell. They can be discriminated by their size and biogenesis into the subsets of apoptotic bodies (diameter > 1μm), microvesicles (diameter range = 1 - 0.1 μm) and exosomes (diameter < 0.1 μm). While microvesicles and apoptotic bodies are shedded from the plasma membrane, exosomes originate from intracellular located multivesicular bodies, which have to fuse with the plasma membrane for their release into the extracellular space. Crosstalk of EVs from MSCs with immune cells is a secured fact, but the way of up-take into the target cells and especially the mechanism of immune modulation are not entirely understood. In our study, we isolated and characterized EVs from a human mesenchymal cardiac cell type regarding their potential to modulate induced immune responses in vitro. The presence of typical EV surface markers like tetraspanins (CD9, CD63, CD81) was confirmed as well as their low expression of HLA-molecules, which indicates a general low immunogenicity. Furthermore, EVs were able to attenuate triggered T cell proliferation accompanied by significantly reduced levels of pro-inflammatory cytokines (IFNγ, TNFα), which was highly dependent on the presence of CD14-positive cells. Interestingly, the EVs of cardiac mesenchymal cells induced a changed phenotype on these myeloid cells; most prominently a reduced HLA-DR and CD86 expression, but enhanced levels for CD206 and PD-L1. Future studies have to identify key molecular pathways involved in EVs` crosstalk with immune cells and to estimate the benefits but also the risks of this new therapeutic based on mesenchymal cells.

Mitochondrial dysfunction is a key feature of injury to numerous tissues and stem cell aging. Although the tissue regenerative role of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) is well known, their specific role in regulating mitochondrial function in target cells remains elusive. Here, we report that MSC-EVs attenuated mtDNA damage and inflammation after acute kidney injury (AKI) and that this effect was at least partially dependent on the mitochondrial transcription factor A (TFAM) pathway. In detail, TFAM and mtDNA were depleted by oxidative stress in MSCs from aged or diabetic donors. Higher levels of TFAM mRNA and mtDNA were detected in normal control (NC) MSC-EVs than in TFAM-knockdown (TFAM-KD) and aged EVs. EV-mediated TFAM mRNA transfer in recipient cells was unaffected by transcriptional inhibition. Accordingly, the application of MSC-EVs restored TFAM protein and TFAM-mtDNA complex (nucleoid) stability, thereby reversing mtDNA deletion and mitochondrial oxidative phosphorylation (OXPHOS) defects in injured renal tubular cells. Loss of TFAM also led to downregulation of multiple anti-inflammatory miRNAs and proteins in MSC-EVs. In vivo, intravenously injected EVs primarily accumulated in the liver, kidney, spleen, and lung. MSC-EVs attenuated renal lesion formation, mitochondrial damage, and inflammation in mice with AKI, whereas EVs from TFAM-KD or aged MSCs resulted in poor therapeutic outcomes. Moreover, TFAM overexpression (TFAM-OE) improved the rescue effect of MSC-EVs on mitochondrial damage and inflammation to some extent. This study suggests that MSC-EVs are promising nanotherapeutics for diseases characterized by mitochondrial damage, and TFAM signaling is essential for maintaining their regenerative capacity.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs.Methods: To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aβ) and glial fibrillary acidic protein (GFAP) levels.Results: The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aβ plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aβ plaques was found in the brain of EV-treated mice compared to saline.Conclusions: Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis.

Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.