Towards an understanding of physical activity-induced post-exertional malaise: Insights into microvascular alterations and immunometabolic interactions in post-COVID condition and myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: A considerable number of patients who contracted SARS-CoV-2 are affected by persistent multi-systemic symptoms, referred to as Post-COVID Condition (PCC). Post-exertional malaise (PEM) has been recognized as one of the most frequent manifestations of PCC and is a diagnostic criterion of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Yet, its underlying pathomechanisms remain poorly elucidated. PURPOSE AND METHODS: In this review, we describe current evidence indicating that key pathophysiological features of PCC and ME/CFS are involved in physical activity-induced PEM. RESULTS: Upon physical activity, affected patients exhibit a reduced systemic oxygen extraction and oxidative phosphorylation capacity. Accumulating evidence suggests that these are mediated by dysfunctions in mitochondrial capacities and microcirculation that are maintained by latent immune activation, conjointly impairing peripheral bioenergetics. Aggravating deficits in tissue perfusion and oxygen utilization during activities cause exertional intolerance that are frequently accompanied by tachycardia, dyspnea, early cessation of activity and elicit downstream metabolic effects. The accumulation of molecules such as lactate, reactive oxygen species or prostaglandins might trigger local and systemic immune activation. Subsequent intensification of bioenergetic inflexibilities, muscular ionic disturbances and modulation of central nervous system functions can lead to an exacerbation of existing pathologies and symptoms.

Endothelial dysfunction and altered endothelial biomarkers in patients with post-COVID-19 syndrome and chronic fatigue syndrome (ME/CFS).

BACKGROUND: Fatigue, exertion intolerance and post-exertional malaise are among the most frequent symptoms of Post-COVID Syndrome (PCS), with a subset of patients fulfilling criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). As SARS-CoV-2 infects endothelial cells, causing endotheliitis and damaging the endothelium, we investigated endothelial dysfunction (ED) and endothelial biomarkers in patients with PCS. METHODS: We studied the endothelial function in 30 PCS patients with persistent fatigue and exertion intolerance as well as in 15 age- and sex matched seronegative healthy controls (HCs). 14 patients fulfilled the diagnostic criteria for ME/CFS. The other patients were considered to have PCS. Peripheral endothelial function was assessed by the reactive hyperaemia index (RHI) using peripheral arterial tonometry (PAT) in patients and HCs. In a larger cohort of patients and HCs, including post-COVID reconvalescents (PCHCs), Endothelin-1 (ET-1), Angiopoietin-2 (Ang-2), Endocan (ESM-1), IL-8, Angiotensin-Converting Enzyme (ACE) and ACE2 were analysed as endothelial biomarkers. RESULTS: Five of the 14 post-COVID ME/CFS patients and five of the 16 PCS patients showed ED defined by a diminished RHI (< 1.67), but none of HCs exhibited this finding. A paradoxical positive correlation of RHI with age, blood pressure and BMI was found in PCS but not ME/CFS patients. The ET-1 concentration was significantly elevated in both ME/CFS and PCS patients compared to HCs and PCHCs. The serum Ang-2 concentration was lower in both PCS patients and PCHCs compared to HCs. CONCLUSION: A subset of PCS patients display evidence for ED shown by a diminished RHI and altered endothelial biomarkers. Different associations of the RHI with clinical parameters as well as varying biomarker profiles may suggest distinct pathomechanisms among patient subgroups.

The TL1A-DR3 Axis Selectively Drives Effector Functions in Human MAIT Cells.

Mucosal-associated invariant T (MAIT) cells are semi-invariant T cells specifically recognizing riboflavin derivatives that are synthesized by many bacteria and fungi presented by MHC class I-related MR1 molecules. Accumulating evidence, however, indicates that MAIT cell functions are inducible by cytokine stimuli in the absence of TCR ligation, identifying MAIT cells as innate sentinels in inflammatory environments. In this study, we demonstrate that death receptor 3 (DR3), a member of the TNFR superfamily, is ex vivo expressed and predominantly upregulated on the surface of human MAIT cells by innate cytokine stimulation. In turn, the DR3 ligand TNF-like protein 1A (TL1A) licenses innate TNF-α production in the absence of cognate triggers, being sufficient to promote activation of primary endothelial cells in vitro. TL1A further amplifies synthesis of IFN-γ and granzyme B in the presence of otherwise weak innate stimuli and strongly augments polyfunctionality. Mechanistically, TL1A potentiates T-bet expression, early NF-κB, and late p38 MAP kinase phosphorylation, with the latter being indispensable for TNF-α production by MAIT cells. Of note, endogenous TL1A is also rapidly released from PBMC cultures in response to bacterial triggering, thereby equally augmenting Ag-specific MAIT cell effector functions. In summary, to our knowledge, we identify a new inflammatory mechanism in MAIT cells linking the DR3/TL1A axis with amplification of TCR-dependent and -independent effector functions, particularly inducing excessive innate TNF-α production. Given that both TL1A and TNF-α are abundantly present at sites of chronic inflammation, the contribution of MAIT cells in such scenarios needs to be determined.

Extracellular vesicles from regenerative human cardiac cells act as potent immune modulators by priming monocytes.

BACKGROUND: Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features. RESULTS: Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1ß) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFß. Further investigations identified CD14+ cells as major recipient cell subset of CardAP-EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells. CONCLUSIONS: CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.

Cardiac Extracellular Vesicles (EVs) Released in the Presence or Absence of Inflammatory Cues Support Angiogenesis in Different Manners.

Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV-cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter® miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application.

Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co-Transplantation.

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8+ T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245.

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane.

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications.

Decellularized amniotic membrane attenuates postinfarct left ventricular remodeling.

BACKGROUND: Placenta and amnion have been suggested as sources of juvenile cells and tissues for use in surgical regenerative medicine. We previously determined the impact of amniotic epithelial cells induced to undergo epithelial-to-mesenchymal transition (EMT) on myocardial remodeling processes and now evaluated the effects of naïve and processed amniotic membrane (AM) on postischemic left ventricular (LV) geometry and function. METHODS: Human AM was used in unmodified form (AM), after EMT induction by transforming growth factor ß (EMT-AM), and after decellularization (Decell-AM). After characterization by histology, electron microscopy, splenocyte proliferation assay, and cytokine release, myocardial infarction was induced in 6-8-week old male BALB/c mice by permanent left anterior descending coronary occlusion, and AM patches were sutured to the anterior LV surface (n = 10 per group). Infarcted hearts without AM or sham-operated mice were used as controls (n = 10 each). After 4 weeks, LV pressure-volume curves were recorded using a conductance catheter before the animals were sacrificed and the hearts analyzed by histology. RESULTS: TGF-ß treatment induced EMT-like changes in amniotic epithelial cells but increased AM xenoreactivity in vitro (splenocyte proliferation) and in vivo (CD4+ cell invasion). Moreover, in vitro interleukin-6 release from AM and from cardiac fibroblasts co-incubated with AM was 300- or 100-fold higher than that of interleukin-10, whereas Decell-AM did not release any cytokines. AM- and Decell-AM-treated hearts had smaller infarct size and greater infarct scar thickness than infarct control hearts, but there was no difference in myocardial capillary density or the number of TUNEL positive apoptotic cells. LV contractile function was better in the AM and EMT-AM groups than in infarcted control hearts, but dP/dt max, dP/dt min, stroke work, and cardiac output were best preserved in mice treated with Decell-AM. Volume-based parameters (LV end-systolic and end-diastolic volume as well as LV ejection fraction) did not differ between AM and Decell-AM. CONCLUSIONS: Decellularized AM supports postinfarct ventricular dynamics independent of the actual regeneration processes. As a cell-free approach to support the infarcted heart, this concept warrants further investigation.

Xeno-immunogenicity of ice-free cryopreserved porcine leaflets.

BACKGROUND: Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. METHODS: We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. RESULTS: None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. CONCLUSIONS: IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.

Immunobiology of naïve and genetically modified HLA-class-I-knockdown human embryonic stem cells.

Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESC(KD)). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESC(KD) was 88%-99% reduced. T cell activation after hESC(KD) transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESC(KD) was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESC(KD) was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.

Ischemia-reperfusion injury: beneficial effects of mesenchymal stromal cells.

PURPOSE OF REVIEW: Organ transplantation and other major surgeries are impacted by ischemia-reperfusion injury (IRI). Mesenchymal stromal cells (MSCs) recently became an attractive alternative therapeutic tool to combat IRI. The present review highlights the effects of MSCs in the preclinical animal models of IRI and clinical trials, and explains their potential modes of action based on the pathophysiological IRI cascade. RECENT FINDINGS: The application of MSCs in animal models of IRI show anti-inflammatory and anti-apoptotic effects, particularly for damage to the kidneys, heart and lungs. The mechanism of MSC action remains unclear, but may involve paracrine factors which could include the transfer of microvesicles, RNA or mitochondria. Although few clinical trials have reached completion, adverse effects appear minimal. SUMMARY: MSCs show promise in protecting against IRI-induced damage. They appear to help recovery mainly by affecting the levels of inflammation and apoptosis during the organ repair process. In addition, they may mediate immunomodulatory effects on the innate and adaptive immune processes triggered during reperfusion and reduce fibrosis. Success in preclinical animal models has led to the initiation of ongoing clinical trials.

Pro-Resolving Mediators in Rotator Cuff Disease: How Is the Bursa Involved?

So far, tendon regeneration has mainly been analyzed independent from its adjacent tissues. However, the subacromial bursa in particular appears to influence the local inflammatory milieu in the shoulder. The resolution of local inflammation in the shoulder tissues is essential for tendon regeneration, and specialized pro-resolving mediators (SPMs) play a key role in regulating the resolution of inflammation. Here, we aimed to understand the influence of the bursa on disease-associated processes in neighboring tendon healing. Bursa tissue and bursa-derived cells from patients with intact, moderate and severe rotator cuff disease were investigated for the presence of pro-resolving and inflammatory mediators, as well as their effect on tenocytes and sensitivity to mechanical loading by altering SPM signaling mediators in bursa cells. SPM signal mediators were present in the bursae and altered depending on the severity of rotator cuff disease. SPMs were particularly released from the bursal tissue of patients with rotator cuff disease, and the addition of bursa-released factors to IL-1ß-challenged tenocytes improved tenocyte characteristics. In addition, mechanical loading modulated pro-resolving processes in bursa cells. In particular, pathological high loading (8% strain) increased the expression and secretion of SPM signaling mediators. Overall, this study confirms the importance of bursae in regulating inflammatory processes in adjacent rotator cuff tendons.

Understanding, diagnosing, and treating Myalgic encephalomyelitis/chronic fatigue syndrome - State of the art: Report of the 2nd international meeting at the Charité Fatigue Center.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a devastating disease affecting millions of people worldwide. Due to the 2019 pandemic of coronavirus disease (COVID-19), we are facing a significant increase of ME/CFS prevalence. On May 11th to 12th, 2023, the second international ME/CFS conference of the Charité Fatigue Center was held in Berlin, Germany, focusing on pathomechanisms, diagnosis, and treatment. During the two-day conference, more than 100 researchers from various research fields met on-site and over 700 attendees participated online to discuss the state of the art and novel findings in this field. Key topics from the conference included: the role of the immune system, dysfunction of endothelial and autonomic nervous system, and viral reactivation. Furthermore, there were presentations on innovative diagnostic measures and assessments for this complex disease, cutting-edge treatment approaches, and clinical studies. Despite the increased public attention due to the COVID-19 pandemic, the subsequent rise of Long COVID-19 cases, and the rise of funding opportunities to unravel the pathomechanisms underlying ME/CFS, this severe disease remains highly underresearched. Future adequately funded research efforts are needed to further explore the disease etiology and to identify diagnostic markers and targeted therapies.

Human leukocyte antigen I knockdown human embryonic stem cells induce host ignorance and achieve prolonged xenogeneic survival.

BACKGROUND: Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics. METHODS AND RESULTS: Using various in vivo, nonischemic, hindlimb xenotransplant models (immunocompetent and defined immunodefective mouse strains) as well as human in vitro T-cell and natural killer (NK)-cell assays, we revealed a central role for T cells in mediating hESC rejection. The NK-cell susceptibility of hESC in vivo was found to be low, and the NK response to hESC challenge in vitro was negligible. To reduce the antigenicity of hESC, we successfully generated human leukocyte antigen (HLA) I knockdown cells (hESC(siRNA+IB)) using both HLA I RNA interference (siRNA) and intrabody (IB) technology. HLA I expression was ≈99% reduced after 7 days and remained low for weeks. Cellular immune recognition of these hESC(siRNA+IB) was strongly reduced in both xenogeneic and allogeneic settings. Immune rejection was profoundly mitigated after hESC(siRNA+IB) transplantation into immunocompetent mice, and even long-term graft survival was achieved in one third of the animals without any immunosuppression. The survival benefit of hESC(siRNA+IB) was further confirmed under ischemic conditions in a left anterior descending coronary artery ligation model. CONCLUSIONS: HLA I knockdown hESC(siRNA+IB) provoke T-cell ignorance and experience largely mitigated xenogeneic rejection. By generating hypoantigeneic hESC lines, the generation of acceptable hESC derivatives may become a practical concept and push cell replacement strategies forward.

Serum of Post-COVID-19 Syndrome Patients with or without ME/CFS Differentially Affects Endothelial Cell Function In Vitro.

A proportion of COVID-19 reconvalescent patients develop post-COVID-19 syndrome (PCS) including a subgroup fulfilling diagnostic criteria of Myalgic encephalomyelitis/Chronic Fatigue Syndrome (PCS/CFS). Recently, endothelial dysfunction (ED) has been demonstrated in these patients, but the mechanisms remain elusive. Therefore, we investigated the effects of patients' sera on endothelia cells (ECs) in vitro. PCS (n = 17), PCS/CFS (n = 13), and healthy controls (HC, n = 14) were screened for serum anti-endothelial cell autoantibodies (AECAs) and dysregulated cytokines. Serum-treated ECs were analysed for the induction of activation markers and the release of small molecules by flow cytometry. Moreover, the angiogenic potential of sera was measured in a tube formation assay. While only marginal differences between patient groups were observed for serum cytokines, AECA binding to ECs was significantly increased in PCS/CFS patients. Surprisingly, PCS and PCS/CFS sera reduced surface levels of several EC activation markers. PCS sera enhanced the release of molecules associated with vascular remodelling and significantly promoted angiogenesis in vitro compared to the PCS/CFS and HC groups. Additionally, sera from both patient cohorts induced the release of molecules involved in inhibition of nitric oxide-mediated endothelial relaxation. Overall, PCS and PCS/CFS patients' sera differed in their AECA content and their functional effects on ECs, i.e., secretion profiles and angiogenic potential. We hypothesise a pro-angiogenic effect of PCS sera as a compensatory mechanism to ED which is absent in PCS/CFS patients.

Immunomodulative efficacy of bone marrow-derived mesenchymal stem cells cultured in human platelet lysate.

Human mesenchymal stem cells (hMSCs) are considered to be a promising tool for novel cell-based therapies. Clinical applications in solid organ transplantation were hampered by the dependence on animal serum for hMSCs clinical scale expansion until substitution with human platelet lysate (HPL) became a promising alternative. Therefore we focused on a direct comparison of immunomodulatory properties of hMSCs cultured in HPL or fetal calf serum (FCS). Phenotypic characterization, detection of cytokine secretion and effects on alloantigen- and mitogen-induced lymphocyte proliferation as well as degranulation of cytomegalovirus-specific cytotoxic T cells were applied in potency assays. We demonstrated that HPL-cultured MSCs have comparable immunomodulatory capacities to their FCS-cultured counterparts. The observed immunomodulatory properties include a beneficial inhibitory effect on immune cell proliferation and an unaffected viral T cell immunity. Thus, culturing hMSCs in HPL generates an efficient and safe expansion combined with intriguing immunomodulatory properties making these cells an attractive cell therapeutic tool.

In vivo effect of bone marrow-derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia.

Brain death and prolonged cold ischemia are major contributors to the poorer long-term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors ('marginal organs') are used. Targeting ischemia-reperfusion injury-related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow-derived rat MSCs was tested in co-cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT-PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose-dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro-inflammatory cytokines, chemokines, and intercellular adhesion molecule-1. In addition, fewer antigen-presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet ß-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic ß-cells via the αvß3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

Human mesenchymal stromal cells and derived extracellular vesicles: Translational strategies to increase their proangiogenic potential for the treatment of cardiovascular disease.

Mesenchymal stromal cells (MSCs) offer great potential for the treatment of cardiovascular diseases (CVDs) such as myocardial infarction and heart failure. Studies have revealed that the efficacy of MSCs is mainly attributed to their capacity to secrete numerous trophic factors that promote angiogenesis, inhibit apoptosis, and modulate the immune response. There is growing evidence that MSC-derived extracellular vesicles (EVs) containing a cargo of lipids, proteins, metabolites, and RNAs play a key role in this paracrine mechanism. In particular, encapsulated microRNAs have been identified as important positive regulators of angiogenesis in pathological settings of insufficient blood supply to the heart, thus opening a new path for the treatment of CVD. In the present review, we discuss the current knowledge related to the proangiogenic potential of MSCs and MSC-derived EVs as well as methods to enhance their biological activities for improved cardiac tissue repair. Increasing our understanding of mechanisms supporting angiogenesis will help optimize future approaches to CVD intervention.

MiRNA Profiles of Extracellular Vesicles Secreted by Mesenchymal Stromal Cells-Can They Predict Potential Off-Target Effects?

The cardioprotective properties of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are currently being investigated in preclinical studies. Although microRNAs (miRNAs) encapsulated in EVs have been identified as one component responsible for the cardioprotective effect of MSCs, their potential off-target effects have not been sufficiently characterized. In the present study, we aimed to investigate the miRNA profile of EVs isolated from MSCs that were derived from cord blood (CB) and adipose tissue (AT). The identified miRNAs were then compared to known targets from the literature to discover possible adverse effects prior to clinical use. Our data show that while many cardioprotective miRNAs such as miR-22-3p, miR-26a-5p, miR-29c-3p, and miR-125b-5p were present in CB- and AT-MSC-derived EVs, a large number of known oncogenic and tumor suppressor miRNAs such as miR-16-5p, miR-23a-3p, and miR-191-5p were also detected. These findings highlight the importance of quality assessment for therapeutically applied EV preparations.