Apoptosis In Mesenchymal Stromal Cells Research Articles

Apoptosis in mesenchymal stromal cells activates an immunosuppressive secretome predicting clinical response in Crohn’s disease

Invivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human Tcell proliferation and activation, and chemoattracted monocytes invitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.

CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

IntroductionHuman mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the ‘senescent load’ of MSC products are needed.MethodsEarly and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; β-galactosidase staining (SA-β-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26high and CD26low MSC(AT). The effect of senolytics on the CD26high subpopulation was evaluated in senescent MSC(AT).ResultsLate passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26high MSC(AT) had reduced immunopotency compared to CD26low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26high MSC(AT).ConclusionsDPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26high MSC(AT) have reduced immunopotency and CD26high MSCs are reduced with senolytics.

Assessing the immunosuppressive activity of alginate-encapsulated mesenchymal stromal cells on splenocytes

Mesenchymal stromal cells (MSCs) show immunosuppressive effects both via cell-to-cell contact (direct) with immune cells and by producing paracrine factors and extracellular vesicles (indirect). A key challenge in delivering this therapeutic effect in vivo is retaining the MSCs at the site of injection. One way to address this is by encapsulating the MSCs within suitable biomaterial scaffolds. Here, we assess the immunosuppressive effect of alginate-encapsulated murine MSCs on proliferating murine splenocytes. Our results show that MSCs are able to significantly suppress splenocyte proliferation by ∼50% via the indirect mechanism and almost completely (∼98%) via the direct mechanism. We also show for the first time that MSCs as monolayers on tissue culture plastic or encapsulated within alginate, when physically isolated from the splenocytes via transwells, are able to sustain immunosuppressive activity with repeated exposure to fresh splenocytes, for as long as 9 days. These results indicate the need to identify design strategies to simultaneously deliver both modes of MSC immunosuppression. By designing cell-biomaterial constructs with tailored degradation profiles, we can achieve a more sustained (avoiding MSCs migration and apoptosis) and controlled release of both the paracrine signals and eventually the cells themselves enabling efficient MSC-based immunosuppressive therapies for wound healing.

Short lifespan of syngeneic transplanted MSC is a consequence of in vivo apoptosis and immune cell recruitment in mice

Mesenchymal stromal cells (MSC) are attractive tools for cell-based therapy, yet the mechanisms underlying their migration and survival post-transplantation are unclear. Accumulating evidence indicates that MSC apoptosis modulates both innate and adaptive immune responses which impact on MSC therapeutic effects. Using a dual tracking system, namely the Luciferase expression and VivoTrack680 labelling, and in vivo optical imaging, we investigated the survival and migration of MSC transplanted by various routes (intravenous, subcutaneous, intrapancreatic and intrasplenic) in order to identify the best delivery approach that provides an accumulation of therapeutic cells to the injured pancreas in the non-obese diabetic (NOD) mouse. The results showed that transplanted MSC had limited migration capacity, irrespective of the administration route, and were short-lived with almost total disappearance at 7 days after transplantation. Within one day after transplantation, cells activated hypoxia signalling pathways, followed by Caspase 3-mediated apoptosis. These were subsequently followed by local recruitment of immune cells at the transplantation site, and the engulfment of apoptotic MSC by macrophages. Our results argue for a “hit and die” mechanism of transplanted MSC. Further investigations will elucidate the molecular crosstalk between the inoculated and the host-immune cells.

Detection of apoptosis in mesenchymal stromal cells ‘in vitro’ to predict their efficacy for treating graft versus host disease

Detection of apoptosis in mesenchymal stromal cells ‘in vitro’ to predict their efficacy for treating graft versus host disease

Comparing the Effect of TGF-β Receptor Inhibition on Human Perivascular Mesenchymal Stromal Cells Derived from Endometrium, Bone Marrow and Adipose Tissues.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-β) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31−CD45− MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Is mesenchymal stromal cell apoptosis necessary for their immunomodulatory capacity?

Background & Aim Mesenchymal stromal cells (MSCs) exhibit therapeutic potential in a broad range of disease conditions. However, the lack of clarity around their mechanism of action presents a significant hurdle in their clinical utility. MSCs do not survive intravenous administration and MSC apoptosis can induce immunomodulatory effects, not unlike other apoptotic cell types. Therefore we sought to understand the role of apoptosis in MSC therapy. Methods, Results & Conclusion Using various methods of labelling and tracking MSCs, as well as different MSC/recipient strain combinations, we showed that MSCs underwent caspase 3-dependent apoptosis in the lungs and were efferocytosed by a hierarchy of lung phagocytes. In alveolar macrophages, this process induced transcriptional modification to shut down inflammation. Treatment with pharmacological compounds that selectively inhibit regulators of apoptosis revealed the molecular players involved in MSC apoptosis and survival. By manipulating the apoptotic status of MSCs and assaying their therapeutic efficacy in disease models commonly used to define MSC potency, we were able to address whether apoptosis is necessary in MSC therapy. Our data have significant implications for the development of MSC potency assays and biomanufacturing for clinical translation.

Finding meaning: Potential relationship between critical quality attributes of MSC's and their Clinical efficacy in GvHD

Background & Aim Mesenchymal stromal cells (MSC) display immunomodulatory properties that make them a very attractive tool to treat diseases such as Acute Graft Versus Host Disease (aGVHD). However, although the use of MSC in aGVHD is feasible and safe, its efficacy is completely unpredictable. MSC were first isolated from bone marrow (BM-MSC) but can also be isolated from different tissues such as Wharton's jelly from Umbilical Cord (WJ-MSC). Indeed WJ-MSC have advantages over BM-MSC for standardization of procedures and large-scale production but equivalence of their immunomodulatory activity is not fully demonstrated. This study seeks to elucidate the relationship between the critical quality attributes of BM-MSC and WJ-MSC and the efficacy in the treatment of aGVHD. Methods, Results & Conclusion To obtain and process the cells, an approved IND was followed in our GMP-compliant production facilities using BM-MSC's (PEI 13-081) or WJ-MSC's (PEI 16-017). Retrospective study of patients of four hospitals of Barcelona area with refractory aGVHD treated with MSC produced at Banc de Sang i Teixits between 2017-2019. We have successfully released 13 clinical-grade batches. Cell viability in all of them was >70% and phenotypic profiling resulted in >95% positivity for the expression of CD73, CD90 and CD105 surface markers. Regarding the HLA-DR- marker, although it was expected to be a negative marker on MSCs, we verified that this expression was highly variable especially in BM-MSC in agreement with our previous observations (Figure 1A). The critical quality attributes for each one of the 13 batches (some of the stock batches have not yet been used in patients) and preliminary results from treated patients are summarized in Figure 1B. Interestingly, no differences were found so far between BM-MSC and WJ-MSC in the clinical response of aGVHD treated patients. Although a comprehensive analysis of the characteristics of MSC may still be needed, it is intriguing that, from our preliminary data, in vitro immunomodulatory capacity of WJ-MSC evaluated by measuring the % of inhibition of PBMNC's proliferation shown in potency assays does not correlate with MSC's clinical efficacy. Further work may be needed to understand the mechanisms of apoptosis in clinical grade MSC in line with recent observations made by other authors who have suggested that the response may be mediated by the receptor's ability to induce MCS's apoptosis.

Mesenchymal stromal cells ameliorate acute lung injury induced by LPS mainly through stanniocalcin-2 mediating macrophage polarization.

BackgroundAcute lung injury (ALI) is a devastating syndrome with no effective pharmacological therapies in the clinic. Mesenchymal stromal cells (MSCs) have been demonstrated to promote inflammation resolution and tissue repair in ALI. However, the specific mechanisms of this have not been clearly elucidated. Stanniocalcin-2 (STC2) is a stress-responsive protein that has anti-oxidative properties. Our previous study found that STC2 is a highly expressed stanniocalcin in MSCs, which may be involved in immunomodulatory activities. However, the role of STC2 in MSCs to resolve ALI has never been elucidated.MethodsSpecific shRNA was used to downregulate STC2 in MSCs. We detected ROS, cell apoptosis, and paracrine factors changes in MSCs. STC2-associated antioxidant genes were also investigated by Co-immunoprecipitation (Co-IP) and immunofluorescence. Macrophage (THP1 cells) phenotype transitions were measured by flow cytometry after coculturing with MSCs in vitro. Then, we used MSCs to treat LPS-induced ALI in mice, and assessed injury scores inflammation, and antioxidant activities in the lungs of the mice. Alveolar macrophage (AM) phenotypes and CFSE-labeled MSC apoptosis in collected bronchoalveolar fluids (BALF) were also analyzed by flow cytometry.ResultsAfter the STC2 knockdown, MSCs increased ROS generation and cell apoptosis after PX12 pretreatment. The antioxidant protein Nrf2 was colocalized with STC2 in the nucleus. A lack of STC2 expression in MSCs produced less interleukin 10 (IL10) and blunted macrophage polarization in THP1 cells. Furthermore, in the murine LPS-induced ALI model, the STC2 knockdown counteracted the inflammatory resolution and antioxidative effect of MSCs in the lungs. MSCshSTC2-treated mice had a higher lung injury score than the controls, which may be attributed to diminished AM polarization and increased apoptosis of MSCs in vivo.ConclusionsCollectively, these results suggested that STC2 is essential to the anti-oxidative and anti-inflammation properties of MSCs and could prove to be crucial for stem cell therapies for ALI.

Decitabine promotes apoptosis in mesenchymal stromal cells isolated from patients with myelodysplastic syndromes by inducing reactive oxygen species generation

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells, resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless, the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here, we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P < 0.00085), cleaved caspase (−3, −8, and −9) activity, and mitochondrial membrane potential reduction. Additionally, DAC-induced apoptosis was inhibited by Kp7-6, a FasL/Fas antagonist, indicating a crucial role of FasL/Fas, a cell death receptor, in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore, N-acetyl-L-cysteine (NAC), a widely accepted ROS scavenger, efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P < 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore, ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs, which serves as a crucial factor for mitochondrial membrane potential reduction, and DAC induces cell death prior to FasL/Fas stimulation.

Asprosin improves the survival of mesenchymal stromal cells in myocardial infarction by inhibiting apoptosis via the activated ERK1/2-SOD2 pathway

AimsSeveral adipokines have been proven to improve the therapeutic efficacy of mesenchymal stromal cells (MSCs) when used to treat ischemic heart disease. Asprosin (ASP) is a newly-discovered adipokine. ASP might also predict the severity of coronary pathology. We investigated the role of ASP on MSCs and the effects of ASP-pretreated MSCs on myocardial infarction (MI). Main methodsMSCs were labelled with a lentivirus carrying green fluorescent protein (GFP). For in vivo study, after pretreatment with vehicle or ASP, MSCs were injected into infarcted hearts. Cardiac function and fibrosis were then evaluated 4 weeks after the induction of MI and survival of MSCs evaluated after 1 week. MSCs proliferation and migration were investigated after ASP treatment in vitro. MSCs apoptosis induced by hydrogen peroxide (H2O2) was assessed using flow cytometry. Key findingsCompared to vehicle-pretreated MSCs, ASP-pretreated MSCs significantly improved the left ventricular ejection fraction (LVEF), and inhibited myocardial fibrosis 4 weeks after MI. ASP pretreatment may have promoted homing of transplanted MSCs. In vitro results showed that ASP had no significant effect on MSC proliferation and migration, but protected these cells from H2O2-induced apoptosis. Among 21 molecules associated with antioxidation and cell death, the antioxidant enzyme SOD2 was significantly upregulated by ASP. Furthermore, ASP treatment inhibited H2O2-induced ROS generation and apoptosis via the activated ERK1/2-SOD2 pathway. SignificanceThis is the first evidence that ASP can regulate MSCs function and enhance MSCs therapy for ischemic heart disease. Furthermore, we demonstrate that ASP protects MSCs from oxidative stress-induced apoptosis via the ERK1/2-SOD2 pathway.

S893 APOPTOTIC MESENCHYMAL STROMAL CELLS INDUCE PROSTAGLANDIN E2 IN MONOCYTES: IMPLICATIONS FOR THE MONITORING OF MESENCHYMAL STROMAL CELL ACTIVITY

Background:Human mesenchymal stromal cells (MSCs) therapy has demonstrated convincing efficacy in steroid‐resistant acute graft‐versus‐host disease (GvHD). Recently, it has been shown that MSC apoptosis and efferocytosis are essential to mediate MSC therapeutic effects in GvHD. However, there are currently no molecular markers for monitoring this process in patients.Aims:To identify the potential tools for this, we decided to interrogate the molecular and functional profile of human phagocytes exposed to apoptotic MSCs (ApoMSCs) and examined these molecules in GvHD patients.Methods:CD14+ monocytes were isolated from healthy donors’ blood whilst ApoMSCs were generated by treating MSCs with anti‐Fas antibody. Monocytes co‐cultured with ApoMSCs for 8 hours (ApoMSC‐Mono) were characterized using flow cytometry, ELISAs and CD3 T cell proliferation assay. Selective cyclooxygenase2 (COX2) inhibitor NS‐398 was added during the monocyte‐conditioning to assess the role of COX2/prostaglandin E2 (PGE2) in ApoMSC‐Mono. To validate our findings in GvHD patients, sera from 8 severe steroid‐resistant GvHD patients before and after MSC treatment were collected and analyzed. Patients’ clinical responses to MSCs, defined by at least 1 stage reduction in any GvHD organ after infusion, were assessed 1 week after the infusion. To compare the results across patients, we calculated the change in PGE2 levels before and after MSC infusion using the formula [PGE2 after infusion ‐ PGE2 before infusion]/PGE2 before infusion x 100%].Results:ApoMSC‐Mono inhibited 50% of the T‐cell proliferation in relative to the monocytes or ApoMSCs alone (Figure 1D, n = 4, p &lt; 0.01). Examining their immunological profiles, there was a significant increase of COX2/PGE2 (n = 3, p &lt; 0.05), indoleamine 2,3‐dioxygenase (IDO) (n = 4, p &lt; 0.01) and programmed death‐ligand 1 (PD‐L1) (n = 3, p &lt; 0.01) compared to the monocyte alone (Figure 1A). Using fluorescent‐labelled ApoMSCs, we confirmed that increased expression of COX2, IDO and PD‐L1 was selectively detected in the monocytes efferocytosing ApoMSCs (Figure 1B, n = 3, p &lt; 0.05). Among the molecules identified, we have found that COX2/PGE2 is the key effector of ApoMSC‐induced immunosuppressive monocytes as COX2 inhibitor NS‐398 significantly impaired the immunosuppressive activity of ApoMSC‐Mono (Figure 1D, n = 4, p &lt; 0.01). NS‐398 prevented the induction of PGE2 (n = 4, p &lt; 0.01), IDO (n = 5, p &lt; 0.01) and PD‐L1 (n = 4, p &lt; 0.01) without interfering with efferocytosis (Figure 1C). Because of this, we asked whether PGE2 levels could reflect the MSC immunological activity in GvHD patients receiving MSC treatment. Therefore, the serum level of PGE2 from 8 GvHD patients before and after the MSC treatment were measured. 4 patients achieved a partial response. One of the responder patients received two independent MSC infusions and assessed twice accordingly. 4 patients were non‐responders. Remarkably, we found that patients responding to MSCs exhibited a significant increment of PGE2 levels compared to the clinical non‐responders (Figure 1E, n = 9, p &lt; 0.05).Summary/Conclusion:Overall, this study reveals the ultimate role of efferocytosis‐induced COX2/PGE2 in delivering immunosuppression, strengthening the role of MSCs apoptosis and efferocytosis in driving MSC immunosuppression. It also advocates the measurement of serum PGE2 in GvHD patients receiving MSC treatment to monitor the immunological effects of MSCs.image

In vivo immunosuppression by apoptotic cells as a mechanism of mesenchymal stromal cell therapy

Background & Aim Mesenchymal stromal cells (MSCs) are one of the most clinically studied cell therapy product. The clinical potential of MSCs in a wide range of disease conditions lies in their immunosuppressive and anti-inflammatory effects. However, marketing approval for MSCs has been slow. A significant hurdle is the lack of clarity around how MSCs exert their broad therapeutic effects in vivo, as these cells become undetectable shortly after administration. Therefore we sought to understand how short-lived MSCs mediate therapeutic effects that persist beyond their survival in vivo. Methods, Results & Conclusion We showed that intravenously administered MSCs underwent caspase 3-dependent apoptosis in the lungs and identified the molecular players involved in MSC apoptosis. Using pharmacological compounds that selectively target these regulators of cell death to induce apoptosis in MSCs, we found that apoptotic MSCs elicited immunosuppressive effects in vivo. Our data further showed that phagocytosis of dying MSCs (efferocytosis) by lung phagocytes is a crucial step in MSC immunosuppression. We identified a hierarchy of professional phagocytic cell types capable of taking up MSCs dying in the lungs. In a mouse model of asthma, this process of MSC apoptosis and efferocytosis triggered the transcriptional modification of alveolar macrophages to shut down inflammation. Our study indicates that the host immune response to dying MSCs is key to their therapeutic effects and clarifies an outstanding question in the field. Furthermore, potency assays should take into account how MSC efferocytosis might contribute to therapeutic outcome.

Dissecting the molecular pathways of apoptosis in mesenchymal stromal cell therapy

Background & Aim Due to their immunosuppressive and anti-inflammatory activities, mesenchymal stromal cells (MSCs) have been extensively utilized in clinical trials to treat a wide range of autoimmune and inflammatory diseases. However, the mechanism underlying MSC therapeutic activity remains poorly understood. Our data show that MSCs undergo apoptosis in the lungs and are undetectable shortly after intravenous administration into mice. In support of this finding, a recent study demonstrated that induction of MSC apoptosis by recipient cytotoxic cells is necessary for immunomodulation in graft-versus-host disease. We are therefore interested in elucidating the molecular pathways of apoptosis that underline MSC therapeutic effect. Methods, Results & Conclusion Using pharmacological compounds that selectively inhibit pro-survival proteins to induce apoptosis in MSCs, we show that mouse and human bone marrow (BM) MSCs engage different molecules for survival. In addition, we are genetically manipulating the apoptotic pathways in mouse and human BM MSCs to gain a greater understanding of how these cells exert their therapeutic effects in vivo, an outstanding question in MSC therapy.

Serum from Asthmatic Mice Potentiates the Therapeutic Effects of Mesenchymal Stromal Cells in Experimental Allergic Asthma.

Asthma is a chronic inflammatory disease characterized by airway inflammation and remodeling, which can lead to progressive decline of lung function. Although mesenchymal stromal cells (MSCs) have shown beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been limited. Mounting evidence suggests that prior exposure of MSCs to specific inflammatory stimuli or environments can enhance their immunomodulatory properties. Therefore, we investigated whether stimulating MSCs with bronchoalveolar lavage fluid (BALF) or serum from asthmatic mice could potentiate their therapeutic properties in experimental asthma. In a house dust mite (HDM) extract asthma model in mice, unstimulated, asthmatic BALF‐stimulated, or asthmatic serum‐stimulated MSCs were administered intratracheally 24 hours after the final HDM challenge. Lung mechanics and histology; BALF protein, cellularity, and biomarker levels; and lymph‐node and bone marrow cellularity were assessed. Compared with unstimulated or BALF‐stimulated MSCs, serum‐stimulated MSCs further reduced BALF levels of interleukin (IL)‐4, IL‐13, and eotaxin, total and differential cellularity in BALF, bone marrow and lymph nodes, and collagen fiber content, while increasing BALF IL‐10 levels and improving lung function. Serum stimulation led to higher MSC apoptosis, expression of various mediators (transforming growth factor‐β, interferon‐γ, IL‐10, tumor necrosis factor‐α‐stimulated gene 6 protein, indoleamine 2,3‐dioxygenase‐1, and IL‐1 receptor antagonist), and polarization of macrophages to M2 phenotype. In conclusion, asthmatic serum may be a novel strategy to potentiate therapeutic effects of MSCs in experimental asthma, leading to further reductions in both inflammation and remodeling than can be achieved with unstimulated MSCs. stem cells translational medicine 2019;8:301&312

Metformin induces apoptosis in mesenchymal stromal cells and dampens their therapeutic efficacy in infarcted myocardium

BackgroundCardiovascular complications, especially myocardial infarctions (MIs), are the leading mortality cause in diabetic patients. The transplantation of stem cells into damaged hearts has had considerable success as a treatment for MI, although whether antidiabetic drugs affect the therapeutic efficacy of stem cell transplantation is still unknown. This study aims to understand whether and how metformin, one of the first-line drugs used to treat type 2 diabetes mellitus (T2DM), induces mesenchymal stromal cell (MSC) apoptosis and dampens their cardioprotective effect after transplantation into infarcted hearts.MethodsA mouse MI model was generated via permanent ligation of the left anterior descending (LAD) coronary artery. MSCs with or without metformin treatment were transplanted after MI in diabetic mice. Echocardiography was used to assess cardiac function and determine cardiac remodeling, and TTC staining was performed to evaluate infarction size. A mouse gavage model was performed to evaluate bone marrow MSCs for flow cytometry assay.ResultsMetformin dampened MSC therapeutic efficacy, which increased infarct size and restricted functional cardiac recovery. Specifically, metformin induced the activation of AMP-activated protein kinase (AMPK)-mediated apoptosis through the inhibition of S6K1-Bad-Bcl-xL cell survival signaling, resulting in the upregulated expression of apoptosis-associated proteins and increased MSC apoptosis. Accordingly, counteracting AMPK attenuated metformin-induced apoptosis in MSCs and partially restored their cardioprotective effects in diabetic mice with MI. Furthermore, a decrease in peripheral blood MSCs was found in patients with T2DM who had a metformin medication history.ConclusionsOur results highlight an unexpected adverse effect of metformin-induced MSC apoptosis through AMPK-mediated mTOR suppression, which is attenuated by an AMPK inhibitor. Moreover, AMPK inhibition may be a novel strategy for enhancing the effectiveness of stem cell therapy after MI in diabetes.

MSC sacrifice for immunosuppression

Transplantation Transfer of mesenchymal stromal cells (MSCs) induces immunosuppression, even though the cells are undetectable shortly after transfer. The immunosuppressive mechanism of MSCs has been something of a mystery. Galleu et al. now suggest that MSC apoptosis is crucial. In a mouse model of graft versus host disease, cytotoxic cells rendered the MSCs apoptotic shortly after transfer. Moreover, cells from patients that responded to MSC therapy had more cytotoxic activity against MSCs. These findings suggest that patients could be screened for likely responsiveness to MSC therapy before transfer. Sci. Transl. Med. 9 , eaam7828 (2017).

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.

The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs.

PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine–induced apoptosis: Potential therapeutic strategy in Parkinson's disease

Background aimsStem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress–induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. MethodsWe transiently transfected Wharton's jelly–derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. ResultsAfter 12–24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40–50%), decreased mitochondrial membrane potential (ΔΨm, ~35–45%), DNA fragmentation (18–30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). Discussion6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.

The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review.

Mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering and regenerative medicine. The multipotent stem cell component of MSC isolates is able to differentiate into derivatives of the mesodermal lineage including adipocytes, osteocytes, chondrocytes, and myocytes. Many common pathways have been described in the regulation of adipogenesis and osteogenesis. However, stimulation of osteogenesis appears to suppress adipogenesis and vice-versa. Increasing evidence implicates a tight regulation of these processes by reactive oxygen species (ROS). ROS are short-lived oxygen-containing molecules that display high chemical reactivity toward DNA, RNA, proteins, and lipids. Mitochondrial complexes I and III, and the NADPH oxidase isoform NOX4 are major sources of ROS production during MSC differentiation. ROS are thought to interact with several pathways that affect the transcription machinery required for MSC differentiation including the Wnt, Hedgehog, and FOXO signaling cascades. On the other hand, elevated levels of ROS, defined as oxidative stress, lead to arrest of the MSC cell cycle and apoptosis. Tightly regulated levels of ROS are therefore critical for MSC terminal differentiation, although the precise sources, localization, levels and the exact species of ROS implicated remain to be determined. This review provides a detailed overview of the influence of ROS on adipogenic and osteogenic differentiation in MSCs.