Stem Cell Derived Extracellular Vesicle Therapy for Multiple Sclerosis, A Systematic Review and Meta-Analysis of Preclinical Studies.

Stem cell therapy holds promise for multiple sclerosis (MS), with efficacy of different stem cell types reported across a range of preclinical MS animal models. While stem cell therapy has been approved for a small number of diseases in humans, extracellular vesicles (EVs) may provide an efficacious, cost-effective, and safer alternative to stem cell therapy. To this end, we conducted a systematic review with meta-analysis to assess the effectiveness of stem cell-derived secretome (EV and conditioned media (CM)) in animal models of MS. The data were extracted to calculate standardized mean differences for primary outcome measure of disease severity, using a random effect model. Additionally, several subgroup analyses were conducted to assess the impact of various study variables such as stem cell type and source, stem cell modification, route and time of administration, number of animals and animal's age, and EV isolation methods on secondary outcome. Publication quality and risk of bias were assessed. Overall, 19 preclinical studies were included in the meta-analysis where stem cell EV/CM was found to significantly reduce disease severity in EV-treated (SMD = 2, 95% CI: 1.18-2.83, P < .00001) and CM-treated animals (SMD = 2.58, 95% CI: 1.34-3.83, P < .00001) compared with controls. Our analysis indicated that stem cell secretome has a positive effect on reducing demyelination, systemic neuroinflammation, and disease severity in preclinical models of MS. These findings indicate a potential therapeutic effect that merits investigation and validation in clinical settings.

Genomic insights into isolation of the threatened Florida crested caracara (Caracara plancus).

We conducted a population genomic study of the crested caracara (Caracara plancus) using samples (n = 290) collected from individuals in Florida, Texas, and Arizona, United States. Crested caracaras are non-migratory raptors ranging from the southern tip of South America to the southern United States, including a federally protected relict population in Florida long thought to have been isolated since the last ice age. Our objectives were to evaluate genetic diversity and population structure of Florida's apparently isolated population and to evaluate taxonomic relationships of crested caracaras at the northern edge of their range. Using DNA purified from blood samples, we conducted double-digest restriction site associated DNA sequencing and sequenced the mitochondrial ND2 gene. Analyses of population structure using over 9,000 SNPs suggest that two major clusters are best supported, one cluster including only Florida individuals and the other cluster including Arizona and Texas individuals. Both SNPs and mitochondrial haplotypes reveal the Florida population to be highly differentiated genetically from Arizona and Texas populations, whereas, Arizona and Texas populations are moderately differentiated from each other. The Florida population's mitochondrial haplotypes form a separate monophyletic group, while Arizona and Texas populations share mitochondrial haplotypes. Results of this study provide substantial genetic evidence that Florida's crested caracaras have experienced long-term isolation from caracaras in Arizona and Texas and thus, represent a distinct evolutionary lineage possibly warranting distinction as an Evolutionarily Significant Unit (ESU) or subspecies. This study will inform conservation strategies focused on long-term survival of Florida's distinct, panmictic population.

An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans.

The tetrameric cytoplasmic FeFe hydrogenase Hnd from Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans) catalyses H2 oxidation and couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin by using a flavin-based electron-bifurcating mechanism. Regarding its implication in the bacterial physiology, we previously showed that Hnd, which is non-essential when bacteria grow fermentatively on pyruvate, is involved in ethanol metabolism. Under these conditions, it consumes H2 to produce reducing equivalents for ethanol production as a fermentative product. In this study, the approach implemented was to compare the two S. fructosivorans WT and the hndD deletion mutant strains when grown on ethanol as the sole carbon and energy source. Based on the determination of bacterial growth, metabolite consumption and production, gene expression followed by RT-q-PCR, and Hnd protein level followed by mass spectrometry, our results confirm the role of Hnd hydrogenase in the ethanol metabolism and furthermore uncover for the first time an essential function for a Desulfovibrio hydrogenase. Hnd is unequivocally required for S. fructosivorans growth on ethanol, and we propose that it produces H2 from NADH and reduced ferredoxin generated by an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase catalyzing the conversion of ethanol into acetate. The produced H2 could then be recycled and used for sulfate reduction. Hnd is thus a reversible hydrogenase that operates in H2-consumption by an electron-bifurcating mechanism during pyruvate fermentation and in H2-production by an electron-confurcating mechanism when the bacterium uses ethanol as electron donor.

NMR-based metabolomic analysis of the physiological role of the electron-bifurcating FeFe-hydrogenase Hnd in Solidesulfovibrio fructosivorans under pyruvate fermentation.

Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans), an anaerobic sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of hydrogen gas (H2) into protons and electrons. One of these, named Hnd, was demonstrated to be an electron-bifurcating hydrogenase Hnd (Kpebe et al., 2018). It couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 and whose function has been recently shown to be involved in ethanol production under pyruvate fermentation (Payne 2022). To understand further the physiological role of Hnd in S. fructosivorans, we compared the mutant deleted of part of the hnd gene with the wild-type strain grown on pyruvate without sulfate using NMR-based metabolomics. Our results confirm that Hnd is profoundly involved in ethanol metabolism, but also indirectly intervenes in global carbon metabolism and additional metabolic processes such as the biosynthesis of branched-chain amino acids. We also highlight the metabolic reprogramming induced by the deletion of hndD that leads to the upregulation of several NADP-dependent pathways.

Secondary Lymphoid Organs in Mesenchymal Stromal Cell Therapy: More Than Just a Filter.

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in inflammatory models of human disease. However, clinical translation has fallen short of expectations, with many trials failing to meet primary endpoints. Failure to fully understand their mechanisms of action is a key factor contributing to the lack of successful commercialisation. Indeed, it remains unclear how the long-ranging immunomodulatory effects of MSCs can be attributed to their secretome, when MSCs undergo apoptosis in the lung shortly after intravenous infusion. Their apoptotic fate suggests that efficacy is not based solely on their viable properties, but also on the immune response to dying MSCs. The secondary lymphoid organs (SLOs) orchestrate immune responses and play a key role in immune regulation. In this review, we will discuss how apoptotic cells can modify immune responses and highlight the importance of MSC-immune cell interactions in SLOs for therapeutic outcomes.

The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate.

Desulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 . It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD+ -reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolite production and consumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism.

Exploiting the preferential phagocytic uptake of nanoparticle-antigen conjugates for the effective treatment of autoimmunity.

Tolerance induction is central to the suppression of autoimmunity. Here, we engineered the preferential uptake of nano-conjugated autoantigens by spleen-resident macrophages to re-introduce self-tolerance and suppress autoimmunity. The brain autoantigen, myelin oligodendrocyte glycoprotein (MOG), was conjugated to 200 or 500 nm silica nanoparticles (SNP) and delivered to the spleen and liver-resident macrophages of experimental autoimmune encephalomyelitis (EAE) mice, used as a model of multiple sclerosis. MOG-SNP conjugates significantly reduced signs of EAE at a very low dose (50 µg) compared to the higher dose (>800 µg) of free-MOG. This was associated with reduced proliferation of splenocytes and pro-inflammatory cytokines secretion, decreased spinal cord inflammation, demyelination and axonal damage. Notably, biodegradable porous SNP showed an enhanced disease suppression assisted by elevated levels of regulatory T cells and programmed-death ligands (PD-L1/2) in splenic and lymph node cells. Our results demonstrate that targeting nano-conjugated autoantigens to tissue-resident macrophages in lymphoid organs can effectively suppress autoimmunity.

Mesenchymal stromal cell apoptosis is required for their therapeutic function.

Multipotent mesenchymal stromal cells (MSCs) ameliorate a wide range of diseases in preclinical models, but the lack of clarity around their mechanisms of action has impeded their clinical utility. The therapeutic effects of MSCs are often attributed to bioactive molecules secreted by viable MSCs. However, we found that MSCs underwent apoptosis in the lung after intravenous administration, even in the absence of host cytotoxic or alloreactive cells. Deletion of the apoptotic effectors BAK and BAX prevented MSC death and attenuated their immunosuppressive effects in disease models used to define MSC potency. Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies.

In Vitro Suppression of T Cell Proliferation Is a Conserved Function of Primary and Immortalized Human Cancer-Associated Fibroblasts.

T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.

Novel Circoviruses Detected in Feces of Sonoran Felids.

Sonoran felids are threatened by drought and habitat fragmentation. Vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. However, little is known about the diversity of viruses present in these populations. Small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. We used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat (Lynx rufus) and puma (Puma concolor) scats collected in Sonora, Mexico. Given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. Three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. Phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately 70% and 63%, respectively. At this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. Further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans.

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

Redirecting adult mesenchymal stromal cells to the brain: a new approach for treating CNS autoimmunity and neuroinflammation?

Mesenchymal stromal cells or stem cells (MSCs) have been shown to participate in tissue repair and are immunomodulatory in neuropathological settings. Given this, their potential use in developing a new generation of personalized therapies for autoimmune and inflammatory diseases of the central nervous system (CNS) will be explored. To effectively exert these effector functions, MSCs must first gain entry into damaged neural tissues, a process that has been demonstrated to be a limiting factor in their therapeutic efficacy. In this review, we discuss approaches to maximize the therapeutic efficacy of MSCs by altering their intrinsic trafficking programs to effectively enter neuropathological sites. To this end, we explore the significant role of chemokine receptors and adhesion molecules in directing cellular traffic to the inflamed CNS and the capacity of MSCs to adopt these molecular mechanisms to gain entry to this site. We postulate that understanding and exploiting these migratory mechanisms may be key to the development of cell-based therapies tailored to respond to the migratory cues unique to the nature and stage of progression of individual CNS disorders.

Optimization Techniques for miRNA Expression in Low Frequency Immune Cell Populations.

In this chapter we outline a RNA extraction method for very low immune cell populations isolated from the central nervous system of mice undergoing experimental autoimmune encephalomyelitis. We compare various normalization and quantification techniques to examine miRNA expression. Our data highlight that employing a mean normalization procedure using a number of well-selected housekeeping miRNA genes, followed by absolute quantification with a standard curve generated from a commercial miRNA oligo, gave the most robust and reproducible miRNA expression results.

Essential role for CCR6 in certain inflammatory diseases demonstrated using specific antagonist and knockin mice.

The chemokine receptor CCR6 marks subsets of T cells and innate lymphoid cells that produce IL-17 and IL-22, and as such may play a role in the recruitment of these cells to certain inflammatory sites. However, the precise role of CCR6 has been controversial, in part because no effective monoclonal antibody (mAb) inhibitors against this receptor exist for use in mouse models of inflammation. We circumvented this problem using transgenic mice expressing human CCR6 (hCCR6) under control of its native promoter (hCCR6-Tg/mCCR6-/-). We also developed a fully humanized mAb against hCCR6 with antagonistic activity. The expression pattern of hCCR6 in hCCR6-Tg/mCCR6-/- mice was consistent with the pattern observed in humans. In mouse models of experimental autoimmune encephalomyelitis (EAE) and psoriasis, treatment with anti-hCCR6 mAb was remarkably effective in both preventive and therapeutic regimens. For instance, in the imiquimod model of psoriasis, anti-CCR6 completely abolished all signs of inflammation. Moreover, anti-hCCR6 attenuated clinical symptoms of myelin oligodendrocyte glycoprotein-induced (MOG-induced) EAE and reduced infiltration of inflammatory cells in the central nervous system. CCR6 plays a critical role in Th17 type inflammatory reactions, and CCR6 inhibition may offer an alternative approach for the treatment of these lesions.

B cell-derived transforming growth factor-ß1 expression limits the induction phase of autoimmune neuroinflammation.

Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-ß1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-ß1 expression in B cells (B-TGF-ß1-/-) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B-TGF-ß1-/- mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B-TGF-ß1-/- mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-ß1-deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-ß1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-ß1, findings that may be relevant to B cell-targeted therapies.

Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.

In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders.

Neuroprotective Potential of Mesenchymal Stem Cell-Based Therapy in Acute Stages of TNBS-Induced Colitis in Guinea-Pigs.

BACKGROUND & AIMS: The therapeutic benefits of mesenchymal stem cells (MSCs), such as homing ability, multipotent differentiation capacity and secretion of soluble bioactive factors which exert neuroprotective, anti-inflammatory and immunomodulatory properties, have been attributed to attenuation of autoimmune, inflammatory and neurodegenerative disorders. In this study, we aimed to determine the earliest time point at which locally administered MSC-based therapies avert enteric neuronal loss and damage associated with intestinal inflammation in the guinea-pig model of colitis. METHODS: At 3 hours after induction of colitis by 2,4,6-trinitrobenzene-sulfonate (TNBS), guinea-pigs received either human bone marrow-derived MSCs, conditioned medium (CM), or unconditioned medium by enema into the colon. Colon tissues were collected 6, 24 and 72 hours after administration of TNBS. Effects on body weight, gross morphological damage, immune cell infiltration and myenteric neurons were evaluated. RT-PCR, flow cytometry and antibody array kit were used to identify neurotrophic and neuroprotective factors released by MSCs. RESULTS: MSC and CM treatments prevented body weight loss, reduced infiltration of leukocytes into the colon wall and the myenteric plexus, facilitated repair of damaged tissue and nerve fibers, averted myenteric neuronal loss, as well as changes in neuronal subpopulations. The neuroprotective effects of MSC and CM treatments were observed as early as 24 hours after induction of inflammation even though the inflammatory reaction at the level of the myenteric ganglia had not completely subsided. Substantial number of neurotrophic and neuroprotective factors released by MSCs was identified in their secretome. CONCLUSION: MSC-based therapies applied at the acute stages of TNBS-induced colitis start exerting their neuroprotective effects towards enteric neurons by 24 hours post treatment. The neuroprotective efficacy of MSC-based therapies can be exerted independently to their anti-inflammatory effects.

Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice. METHODS: Using flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals. RESULTS: In vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed. CONCLUSION: The therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.

Combination therapy of mesenchymal stem cells and serelaxin effectively attenuates renal fibrosis in obstructive nephropathy.

Chronic kidney disease (CKD) results from the development of fibrosis, ultimately leading to end-stage renal disease (ESRD). Although human bone marrow-derived mesenchymal stem cells (MSCs) can accelerate renal repair following acute injury, the establishment of fibrosis during CKD may affect their potential to influence regeneration capacity. Here we tested the novel combination of MSCs with the antifibrotic serelaxin to repair and protect the kidney 7 d post-unilateral ureteral obstruction (UUO), when fibrosis is established. Male C57BL6 mice were sham-operated or UUO-inured (n = 4-6) and received vehicle, MSCs (1 × 10(6)), serelaxin (0.5 mg/kg per d), or the combination of both. In vivo tracing studies with luciferin/enhanced green fluorescent protein (eGFP)-tagged MSCs showed specific localization in the obstructed kidney where they remained for 36 h. Combination therapy conferred significant protection from UUO-induced fibrosis, as indicated by hydroxyproline analysis (P < 0.001 vs. vehicle, P < 0.05 vs. MSC or serelaxin alone). This was accompanied by preserved structural architecture, decreased tubular epithelial injury (P < 0.01 vs. MSCs alone), macrophage infiltration, and myofibroblast localization in the kidney (both P < 0.01 vs. vehicle). Combination therapy also stimulated matrix metalloproteinase (MMP)-2 activity over either treatment alone (P < 0.05 vs. either treatment alone). These results suggest that the presence of an antifibrotic in conjunction with MSCs ameliorates established kidney fibrosis and augments tissue repair to a greater extent than either treatment alone.