Preconditioned Mesenchymal Stem Cells Research Articles

THE THERAPEUTIC POTENTIAL OF PRECONDITIONED MESENCHYMAL STEM CELLS IN ATHEROSCLEROSIS

Atherosclerosis (AS) is a multifactorial and inflammatory disease the cause of which is the formation of lipid and cholesterol plaques on the inner wall of blood vessels. Experimental and clinical studies have shown that the pathogenesis of atherosclerosis is, in particular, connected with inflammatory reactions. Recent studies reported that preconditioned mesenchymal stem cells (MSCs) possess an enhanced immunomodulatory potential. The purpose of the study was to investigate the therapeutic potential of preconditioned MSCs in the development of atherosclerosis. MSCs were isolated from the compact bone of mice by standard protocol. The characterization was conducted using flow cytometry, CFU assay and three-lineage differentiation. Cells were preconditioned with different combinations of cytokines and the optimal concentrations were determined by ELISA and by analysis of co-culturing with lymphocytes. The cytokine-preconditioned MSCs were administrated to mice with induced atherosclerosis. The lipids analysis, measuring of mouse spleens’ weights, ELISA and histological analysis of the aorta sections were done after 8-th weeks. We isolated MSCs and then confirmed the possession of the immunomodulatory properties. Preconditioning with the TNF-α cytokine effectively increased the immunomodulatory properties of MSCs compared to untreated MSCs in vitro. The results of in vivo study that the use of TNF-α-preconditioned MSCs reduced the development of atherosclerosis in mice compared to untreated MSCs: it increased the level of Treg cells in the spleen of mice; significantly reduced the levels of TNF-α and IFN-γ in blood serum; significantly reduced the spleen weights of mice; slightly decreased the total cholesterol levels and also increased HDL levels. Administration of TNF-α-preconditioned MSCs reduced the size of atherosclerotic plaques in the aorta of mice. The results of this study demonstrated the effect of cytokine-preconditioned MSCs in the inhibition of the development of atherosclerosis in a mouse model. However, for application as a therapy for suffered patients additional studies are needed. Besides, using of TNF-α preconditioned MSCs can be considered in other preclinical studies of the different diseases.

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

Nisin-preconditioned mesenchymal stem cells combatting nosocomial Pseudomonas infections

BackgroundNosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa are a considerable public health threat, requiring innovative therapeutic approaches. ObjectivesThis study explored preconditioning mesenchymal stem cells (MSCs) with the antimicrobial peptide Nisin to enhance their antibacterial properties while maintaining regenerative capacity. MethodsHuman MSCs were preconditioned with varying concentrations of Nisin (0.1–1000 IU/mL) to determine an optimal dose. MSCs preconditioned with Nisin were characterized using microscopy, flow cytometry, gene expression analysis, and functional assays. The effects of preconditioning on the viability, phenotype, differentiation capacity, antimicrobial peptide expression, and antibacterial activity of MSCs against Pseudomonas aeruginosa were tested in vitro. The therapeutic efficacy was evaluated by topically applying conditioned media from Nisin-preconditioned versus control MSCs to infected wounds in a rat model, assessing bacterial burden, healing, host response, and survival. ResultsAn optimal Nisin dose of 500 IU/mL was identified, which increased MSC antibacterial gene expression and secretome activity without compromising viability or stemness. Nisin-preconditioned MSCs showed upregulated expression of LL37 and hepcidin. Conditioned media from Nisin-preconditioned MSCs exhibited about 4-fold more inhibition of P. aeruginosa growth compared to non-preconditioned MSCs. In the wound infection model, the secretome of Nisin-preconditioned MSCs suppressed bacterial load, accelerated wound closure, modulated inflammation, and improved survival compared to standard MSC treatments. ConclusionThis study explores the effect of preconditioning MSCs with the antimicrobial peptide Nisin on enhancing their antibacterial properties while maintaining regenerative capacity. Secreted factors from Nisin-preconditioned MSCs have the potential to attenuate infections and promote healing in vivo. The approach holds translational promise for managing antibiotic-resistant infections and warrants further development. Preconditioned MSCs with Nisin may offer innovative, multifaceted therapies for combating nosocomial pathogens and promoting tissue regeneration.

Reveal more mechanisms of precondition mesenchymal stem cells inhibiting inflammation.

Hypoxia can get more ability to inhibit inflammation. But how it impact on survival time of mesenchymal stem cells (MSCs) is confusing and how preconditioned MSCs inhibiting inflammation are partially known. Those issues decided the value of preconditioned MSCs by hypoxia.

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo.

Cardiovascular diseases particularly myocardial infarction (MI) are the leading cause of mortality and morbidity around the globe. As cardiac tissue possesses very limited regeneration potential, therefore use of a potent small molecule, inhibitor Wnt production-4 (IWP-4) for stem cell differentiation into cardiomyocytes could be a promising approach for cardiac regeneration. Wnt pathway inhibitors may help stem cells in their fate determination towards cardiomyogenic lineage and provide better homing and survival of cells in vivo. Mesenchymal stem cells (MSCs) derived from the human umbilical cord have the potential to regenerate cardiac tissue, as they are easy to isolate and possess multilineage differentiation capability. IWP-4 may promote the differentiation of MSCs into the cardiac lineage. To evaluate the cardiac differentiation ability of IWP-4 and its subsequent in vivo effects. Umbilical cord tissue of human origin was utilized to isolate the MSCs which were characterized by their morphology, immunophenotyping of surface markers specific to MSCs, as well as by tri-lineage differentiation capability. Cytotoxicity analysis was performed to identify the optimal concentration of IWP-4. MSCs were treated with 5 μM IWP-4 at two different time intervals. Differentiation of MSCs into cardiomyocytes was evaluated at DNA and protein levels. The MI rat model was developed. IWP-4 treated as well as untreated MSCs were implanted in the MI model, then the cardiac function was analyzed via echocardiography. MSCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) dye for tracking, while the regeneration of infarcted myocardium was examined by histology and immunohistochemistry. MSCs were isolated and characterized. Cytotoxicity analysis showed that IWP-4 was non-cytotoxic at 5 μM concentration. Cardiac specific gene and protein expression analyses exhibited more remarkable results in fourteen days treated group that was eventually selected for in vivo transplantation. Cardiac function was restored in the IWP-4 treated group in comparison to the MI group. Immunohistochemical analysis confirmed the homing of pre-differentiated MSCs that were labeled with DiI cell labeling dye. Histological analysis confirmed the significant reduction in fibrotic area, and improved left ventricular wall thickness in IWP-4 treated MSC group. Treatment of MSCs with IWP-4 inhibits Wnt pathway and promotes cardiac differentiation. These pre-conditioned MSCs transplanted in vivo improved cardiac function by cell homing, survival, and differentiation at the infarcted region, increased left ventricular wall thickness, and reduced infarct size.

Hypoxic preconditioned mesenchymal stem cells ameliorate rat brain injury after cardiopulmonary resuscitation by suppressing neuronal pyroptosis.

Cardiac arrest (CA) can result in cerebral ischaemia-reperfusion injury and poor neurological outcomes. While bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have protective effects in brain ischaemic disease, their efficacy can be reduced by the poor oxygen environment. In this study, we investigated the neuroprotective effects of hypoxic preconditioned BMSCs (HP-BMSCs) and normoxic BMSCs (N-BMSCs) in a cardiac arrest rat model by examining their ability to ameliorate cell pyroptosis. The mechanism underlying the process was also explored. Cardiac arrest was induced in rats for 8 min and surviving rats received 1 × 106 normoxic/hypoxic BMSCs or PBS via intracerebroventricular (ICV) transplantation. Neurological function of rats was evaluated using neurological deficit scores (NDSs) and examined for brain pathology. Serum S100B and neuron-specific enolase (NSE) levels and cortical proinflammatory cytokines were measured to evaluate brain injury. Pyroptosis-related proteins in the cortex after cardiopulmonary resuscitation (CPR) were measured using western blotting and immunofluorescent staining. Transplanted BMSCs were tracked using bioluminescence imaging. Results showed significantly better neurological function and neuropathological damage after transplantation with HP-BMSCs. In addition, HP-BMSCs reduced levels of pyroptosis-related proteins in the rat cortex after CPR and significantly reduced levels of biomarkers for brain injury. Mechanistically, HP-BMSCs alleviated brain injury by reducing the expressions of HMGB1, TLR4, NF-κB p65, p38 MAPK and JNK in the cortex. Our study demonstrated that hypoxic preconditioning could enhance the efficacy of BMSCs in alleviating post-resuscitation cortical pyroptosis. This effect may be related to the regulation of the HMGB1/TLR4/NF-κB, MAPK signalling pathways.

Evaluation of immunomodulatory effects of co-culture or supernatant of dexamethasone or IFN-γ-treated adipose-derived mesenchymal stem cells on spleen mononuclear cells

Although mesenchymal stem cells (MSCs) have exhibited promising immunomodulatory potential in preclinical studies, clinical studies have revealed variable results. These results often depend on environmental cues. Pre-conditioning MSCs with cytokines is one of the methods used to enhance their immunomodulatory effects. In this study, we harvested adipose-derived MSCs from mice and cultured them with different doses of the cytokine, IFN-γ, and the corticosteroid drug, dexamethasone, in order to investigate their effects on MSC immunosuppressive function. We found the co-culture or supernatant of MSCs, pre-conditioned with IFN-γ, together with spleen mononuclear cells resulted in a significant reduction of mononuclear cell proliferation. Although the supernatant of MSCs, pre-conditioned with dexamethasone, showed similar results, dexamethasone pre-conditioning of co-cultured MSCs increased mononuclear cell proliferation. The results further our understanding of immune-related effects of MSCs which may provide a basis for further in vivo studies to achieve better clinical results. We propose that pre-conditioning with cytokines might be an effective method to boost the immunomodulatory effects of MSCs.

VEGF Contributes to Mesenchymal Stem Cell-Mediated Reversion of Nor1-Dependent Hypertrophy in iPS Cell-Derived Cardiomyocytes.

Myocardial hypertrophy is present in many heart diseases, representing a strong predictor of adverse cardiovascular outcomes. Regarding therapeutic intervention, mesenchymal stem cells (MSCs) have been suggested to significantly reduce cardiac hypertrophy and progression to heart failure. Preconditioning of MSCs was previously demonstrated to highly improve their paracrine activity resulting in modulation of immune responses and the progression of diseases. Here, we studied the effects of bone marrow-derived preconditioned MSCs on hypertrophied induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) and also sought to identify MSC-derived antihypertrophic molecules. Phenylephrine (PE) was used to induce hypertrophy in murine iPS-CM, and markers of hypertrophy were identified by microarray analysis. Murine MSCs were treated with IFN-γ and IL-1β to enhance their paracrine activity, and transcriptional profiling was performed by microarray analysis. Hypertrophied iPS-CM were subsequently cocultured with preconditioned MSCs or MSC-conditioned medium (CM), respectively. Effects on hypertrophied iPS-CM were studied by cell area quantification, real-time PCR, and western blot. In some experiments, cells were incubated with fractions of MSC-CM obtained by ultrafiltration or by MSC-CM supplemented with inhibitory antibodies. Intracellular and extracellular levels of vascular endothelial growth factor (VEGF) were evaluated by western blot and ELISA. PE-induced hypertrophy in iPS-CM was associated with an upregulation of neuron-derived orphan receptor (Nor1) expression, activation of Akt, and inhibition of both strongly prevented hypertrophy induction in iPS-CM. VEGF secreted by preconditioned MSCs provoked hypertrophy regression in iPS-CM, and a negative correlation between Nor1 expression and hypertrophic growth could be evidenced. Our results demonstrate that Nor1 expression strongly supports hypertrophy in iPS-CM. Moreover, the secretome of preconditioned MSCs triggered regression of hypertrophy in iPS-CM in a VEGF-dependent manner. We suggest that the delivery of the MSC-derived secretome may represent a therapeutic strategy to limit cardiac hypertrophy. However, additional in vivo studies are needed to prove this hypothesis.

Intranasal Administration of Mesenchymal Stem Cell Secretome Reduces Hippocampal Oxidative Stress, Neuroinflammation and Cell Death, Improving the Behavioral Outcome Following Perinatal Asphyxia.

Perinatal Asphyxia (PA) is a leading cause of motor and neuropsychiatric disability associated with sustained oxidative stress, neuroinflammation, and cell death, affecting brain development. Based on a rat model of global PA, we investigated the neuroprotective effect of intranasally administered secretome, derived from human adipose mesenchymal stem cells (MSC-S), preconditioned with either deferoxamine (an hypoxia-mimetic) or TNF-α+IFN-γ (pro-inflammatory cytokines). PA was generated by immersing fetus-containing uterine horns in a water bath at 37 °C for 21 min. Thereafter, 16 μL of MSC-S (containing 6 μg of protein derived from 2 × 105 preconditioned-MSC), or vehicle, were intranasally administered 2 h after birth to asphyxia-exposed and control rats, evaluated at postnatal day (P) 7. Alternatively, pups received a dose of either preconditioned MSC-S or vehicle, both at 2 h and P7, and were evaluated at P14, P30, and P60. The preconditioned MSC-S treatment (i) reversed asphyxia-induced oxidative stress in the hippocampus (oxidized/reduced glutathione); (ii) increased antioxidative Nuclear Erythroid 2-Related Factor 2 (NRF2) translocation; (iii) increased NQO1 antioxidant protein; (iv) reduced neuroinflammation (decreasing nuclearNF-κB/p65 levels and microglial reactivity); (v) decreased cleaved-caspase-3 cell-death; (vi) improved righting reflex, negative geotaxis, cliff aversion, locomotor activity, anxiety, motor coordination, and recognition memory. Overall, the study demonstrates that intranasal administration of preconditioned MSC-S is a novel therapeutic strategy that prevents the long-term effects of perinatal asphyxia.

IGF-1 and HGF Preconditioning of Bone Marrow Mesenchymal Stem Cells Reduced Proteinuria in Rat Focal Segmental Glomerulosclerosis Model

Background & Aim Focal Segmental Glomerulosclerosis (FSGS) is one of the leading causes of end-stage kidney disease requiring renal replacement therapy for survival. It is characterized by massive proteinuria that results in hypoalbuminemia, edema and hyperlipidemia. and is known as irreversible kidney disease that steroid-based drugs only slow down the progression. The use of Mesenchymal stem cells (MSCs) has been successful in the treatment of glomerular diseases. The biggest challenge of the MSC application is its engraftment into the site of injury and its loss due to the poor microenvironment. Preconditioning of MSCs by particular growth factors such as insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) increases their migration, immunomodulation and survival. IGF-1 and HGF have been proposed as promising therapeutic agents in recent years and are especially preferred in renal diseases with their renotrophic and nephroprotective effects. Therefore we aimed to test the effect of IGF-1 and HGF-preconditioned MSCs in a rat model of FSGS. Methods, Results & Conclusion Preconditioning of MSCs was performed by 10 ng/ml IGF-1 and 20 ng/ml HGF and designated as iMSCs. Rat (Sprague-Dawley) FSGS model was technically induced by a single-dose intraperitoneal administration of Puromycin Aminonucleoside (PAN) (150 mg/kg). There were PBS, MSC and iMSC treatment groups (n=6/gr77). The occurrence of FSGS was assessed by biochemistry (i.e. elevated urinary protein/creatinine (P/C) ratio), immunohistochemistry (i.e. decreased glomerular SYNPO protein level) and electron microscopy (i.e. presence of podocyte effacement). We applied 2 × 105 MSC or preconditioned MSC intravenously on the day of 4 following PAN administration. The effect of treatment was assessed by urinary P/C ratios in 24-hour urine which was collected on the treatment day of 9. iMSC treatment significantly decreased proteinuria (p As a result of our study, we demonstrated that iMSCs can partially reverse the glomerular damage and reduce proteinuria with regenerating glomerular filtering apparatus in the PAN model of FSGS and therefore may offer a new therapeutic strategy for human FSGS.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization

BackgroundSpinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism.MethodsElectron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms.ResultsOur results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p.ConclusionHypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Intrarenal Transplantation of Hypoxic Preconditioned Mesenchymal Stem Cells Improves Glomerulonephritis through Anti-Oxidation, Anti-ER Stress, Anti-Inflammation, Anti-Apoptosis, and Anti-Autophagy.

To confer further therapeutic potential and prevent some adverse effects by the mesenchymal stem cells (MSCs) transplantation, we explored the effects of locally intrarenal arterial administration of hypoxic preconditioned MSCs in the anti-Thy1.1 induced rat glomerulonephritis. Proteinuria, histochemical staining, and western blotting were used to explore the therapeutic effects and mechanisms. Locally intrarenal arterial MSCs transplantation successfully implanted the fluorescent or CD44 labeled MSCs in the nephritic glomeruli, ameliorated proteinuria, and glomerulosclerosis in nephritic rats. Hypoxic preconditioning significantly upregulated hypoxic inducible factor-1α/VEGF (HIF-1α/VEGF) in the MSCs and was more efficient than normoxic MSCs in reducing the degree of urinary protein, glomerulosclerosis, fibrosis, macrophage/monocyte infiltration, GRP78 mediated endoplasmic reticulum stress, Beclin-1/LC3-II mediated autophagy, and Bax/Bcl-2/caspase 3 mediated apoptosis. Hypoxic MSCs could further promote intranuclear nuclear factor (erythroid-derived 2, Nrf2) and reduce nuclear factor kappa B expression in nephritic kidneys. As compared to normoxic MSCs, hypoxic MSCs transplantation significantly upregulated the renal expression of anti-oxidative response elements/enzymes including glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione peroxidase, catalase, Mn, and Cu/Zn superoxide dismutase. In summary, intrarenal hypoxic preconditioning MSCs transplantation was more effective to activate hypoxic inducible factor-1α/VEGF/Nrf2 (HIF-1α/VEGF/Nrf2) signaling, preserve anti-oxidant proteins and anti-oxidative responsive element proteins, and subsequently reduce glomerular apoptosis, autophagy, and inflammation.

Hypoxic preconditioning improves the survival and neural effects of transplanted mesenchymal stem cells via CXCL12/CXCR4 signalling in a rat model of cerebral infarction.

The treatment of neural deficiency after cerebral infarction is challenging, with limited therapeutic options. The transplantation of mesenchymal stem cells (MSCs) to the ischemic penumbra is a potential therapeutic approach. In the present study, a cerebral infarction model was generated by performing middle cerebral artery occlusion (MCAO) in SD rats. The expression of CXCR4 increased, and the number of MSCs migrating to the peri-infarct area was higher in rats transplanted with preconditioned MSCs than in rats transplanted with untreated MSCs. The rate of apoptosis, as evaluated by TUNEL staining and immunoblotting assays, was reduced in rats receiving preconditioned MSCs. A significant amelioration of neural regeneration and improved neurological function were observed in rats injected with preconditioned MSCs compared with those injected with untreated MSCs. However, the application of an siRNA targeting CXCL12 significantly inhibited the protective role of preconditioned MSCs against apoptosis and promoted the migration of MSCs to the ischemic area, leading to impaired neuronal regeneration and limited recovery of neuronal function. Hypoxic preconditioning of MSCs prior to transplantation suppressed apoptosis and increased their migration abilities, leading to the promotion of neuronal regeneration and improvement in neural function after transplantation. This preconditioning strategy may be considered as a potential approach for the modification of MSCs prior to cell transplantation therapy in patients with cerebral infarction. SIGNIFICANCE OF THE STUDY: We found that hypoxic preconditioning of MSCs improved their ability to promote neuronal regeneration and the recovery of neuronal function. Moreover, we showed that CXCR4 inhibited apoptosis, improved cell homing, and promoted neuronal differentiation, without influencing angiogenesis. Our study provides a relatively safe preconditioning method for potential use for cell transplantation therapy in ischemic cerebral infarction. The results presented here will facilitate the development of novel strategies and techniques to improve the tolerance and migration ability of transplanted cells for the treatment of cerebral infarction sequelae.

The neuroprotection of hypoxic adipose tissue-derived mesenchymal stem cells in experimental traumatic brain injury

Traumatic brain injury is one of the leading causes of mortality and morbidity worldwide. At present there is no effective treatment. Previous studies have demonstrated that topical application of adipose tissue-derived mesenchymal stem cells can improve functional recovery in experimental traumatic brain injury. In this study, we evaluated whether hypoxic preconditioned mesenchymal stem cells could enhance the recovery from traumatic brain injury. Traumatic brain injury was induced with an electromagnetically controlled cortical impact device. Two million mesenchymal stem cells derived from the adipose tissue of transgenic green fluorescent protein Sprague-Dawley rats were cultured under either hypoxic (2.5% O2 for 18 hours) (N = 30) or normoxic (18% O2) (N = 30) conditions, then topically applied to the exposed cerebral cortex within 1 hour after traumatic brain injury. A thin layer of fibrin was used to fix the cells in position. No treatment was given to the animals with traumatic brain injury (N = 30). Animals that underwent craniectomy without traumatic brain injury were treated as the sham group (N = 15). Neurological functions were evaluated with water maze, Roto-rod and gait analysis. Animals were sacrificed at days 3, 7, and 14 for microscopic examinations and real-time polymerase chain reaction analysis. The rats treated with hypoxic mesenchymal stem cells showed the greatest improvement in neurological function recovery. More green fluorescent protein-positive cells were found in the injured brain parenchyma treated with hypoxic mesenchymal stem cells that co-expressed glial fibrillary acidic protein, Nestin, and NeuN. Moreover, there was early astrocytosis triggered by the infiltration of more glial fibrillary acidic protein-positive cells and microgliosis was suppressed with fewer ionized calcium binding adapter molecule 1-positive cells in the penumbra region of hypoxic mesenchymal stem cells group at day 3. Compared with normoxic mesenchymal stem cells and traumatic brain injury only groups, there was significantly (p < 0.05) less neuronal death in both the hippocampus and penumbral regions in sections treated with hypoxic mesenchymal stem cells as determined by Cresyl violet and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining respectively. The expression of pro-inflammatory genes (interleukin 6, interleukin 1a, interleukin 1b, tumor necrosis factor α) was upregulated and apoptotic gene (Caspase-3) expression was suppressed at day 3. Anti-inflammatory (interleukin 10) and anti-apoptotic (BCL2 associated agonist of cell death) gene expression was upregulated at days 7 and 14. Our study showed that a hypoxic precondition enhanced the beneficial effects of mesenchymal stem cells on neurological recovery after traumatic brain injury.

Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages

Pathogen-associated molecular patterns, damage-associated molecular patterns, and other noxious stimuli activate macrophages to induce the proinflammatory responses. Modulation of inflammatory macrophages (M1) into an anti-inflammatory tissue repair macrophage (M2) phenotype at the appropriate time optimizes bone remodeling and regeneration. Simulating the proinflammatory stimuli by using preconditioned mesenchymal stem cells (MSCs) at an earlier stage, and alleviate the inflammation by using IL4-secreting MSCs at a later stage could further optimize bone regeneration in chronic inflammatory conditions, including periprosthetic osteolysis.

Abstract 339: A Novel Strategy to Harness Stem Cell-Intrinsic Mechanobiological Properties for Advanced Tissue Repair: Implication in Cardiac Tissue Injury

Rationale: Stem cells are constantly exposed to biomechanical and biochemical signals when transplanted in a continuously beating cardiac environment for treatment. Here, we postulated that an in vitro miniaturized beating cardiac microenvironment can induce unique biomechanical stimuli to co-cultured MSCs that can influence therapeutic potential of the later. This project aimed (i) to pre-condition mesenchymal stem cells (MSCs) with continuous mechanobiological stimuli using a 3D cardiac tissue environment (ii) to intramyocardially inject pre-conditioned MSCs into mice heart with myocardial infarction (MI) for tissue repair. Methods: We fabricated an in vitro cardiac platform as shown in Fig 1 , using an electro-conductive hydrogel and human iPSC-derived cardiac cells. Adipose MSCs were then injected to the beating hydrogel platform. After 7 days, activated (MB+) and non-activated MSCs (MB-) were isolated for in vitro and in vivo analysis. Results: The force generated by the constant beating of cardiomyocytes induced profound effects in on MSC behavior in the MB+ group. Several mechano-transduction factors (YAP1, TAZ), actin polymerization genes involved in cardiac muscle contractions ( ACTA2 , CDC42, TNNT2 ) and angiogenic genes were upregulated in MB+. RNA-seq analysis further confirmed these findings. 3 week study in mice with MI demonstrated significant increase in capillary density and reduction in infarct sizes in MB+ (n=6) compared to MB- group. Furthermore, the MB+ group showed significantly higher cardiac performance in echocardiography. We can conclude MB-activated MSCs can have significant impact in future treatment strategies for patients with MI.

Effect of CoCl2 or H2O2 pre-conditioned mesenchymal stem cells in a mouse model of pulmonary fibrosis

Introduction: Pulmonary Fibrosis is characterized by excessive matrix deposition which leads to airway remodeling and disruption of the typical architecture of the lung parenchyma. The disease progression is associated with a high mortality rate. The current treatment for pulmonary fibrosis includes drugs which either reduce progression of the disease or provide symptomatic relief. Multiple studies have examined the effect of cell-based therapy in pulmonary fibrosis. We investigated the effect of administration of pre-conditioned bone marrow-derived mesenchymal stem cells (BMMSC) in a mouse model of pulmonary fibrosis.&#x0D; Methods: Firstly, we examined the effect of pre-conditioning on the cells using cell-based assays. We found that pre-conditioning did not significantly alter cell proliferation or led to cellular inflammation. The cells continued to express MSC marker, CD105, and pluripotency marker Oct3/4. Next, we evaluated the proliferative and anti-inflammatory potential of BMMSC administration using a series of assays in a mouse model of pulmonary fibrosis. Bleomycin was administered to induce pulmonary fibrosis in mice on Day 0. MSCs were administered on day 1 and day 3; the mice were sacrificed on day 22, and their tissues were collected for analysis.&#x0D; Results: We found that similar to untreated cells, administration of pre-conditioned cells resulted in an increase in the proliferative potential and reduction in inflammation in the lung tissue, bronchoalveolar lavage, bone marrow, and blood. We observed reduction in the number of granulocytes in peripheral blood upon MSC administration. However, we did not observe any structural changes in the lung upon MSC administration. We found a small reduction in collagen content in the lung which was also seen upon staining with Masson's trichrome.&#x0D; Conclusion: These results demonstrate that pre-conditioned BM-MSC lead to improvement in the disease state through paracrine effects but pre-conditioning of cells for 24 hours does not significantly improve the beneficial effect of MSC administration.

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome.

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1β were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1β from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.

Mesenchymal Stem Cells Preconditioned with Myeloma Cells from High-Risk Patients Support the Growth of Myeloma Cells from Low-Risk Patients

Introduction:Each disease stage in myeloma (MM) is associated with parallel changes in both the MM clone and the bone marrow (BM) microenvironment. Mesenchymal cell lineages derived from mesenchymal stem cells (MSCs), including osteoblasts, adipocytes and pericytes play an important role in MM cell growth mediated by the modification of the MM niche in the BM.The overall goal of the study was to test and identify changes induced in MSCs by high-risk (HR) MM cells that impact MSC function and promote oncogenic pathways capable of supporting low-risk (LR) MM cells.Methods:Normal MSCs were either cultured alone ("unconditioned") or co-cultured with MM cells for 5 days. The cultured and co-cultured cells were trypsinized, replated for 40 min, followed by serial washing to remove MM cells from the adherent MSCs. More than 95% of the remaining adherent cells after co-culture were MSCs ("preconditioned"). The unconditioned and preconditioned MSCs or their 24 hrs conditioned media (CM; 50%) were tested for their ability to support the 5-days growth of CD138+ MM cells from LR (n=4) and HR (n=3) patients.To identify factors altered in MSCs by HR MM cells, the unconditioned and preconditioned MSCs and their serum-free conditioned media (n=4) underwent gene expression profiling and proteomic analysis. Whole bone biopsy gene expression profiles from newly diagnosed patients with MM enrolled in Total Therapy clinical trials were used to correlate the altered expression of factors in preconditioned MSCs with their expression in clinical samples.Results:Growth of all MM cells tested was increased by inclusion of MSCs preconditioned with HR MM cells by 2.2± 0.2 (p<0.0004) and by CM from these MSCs by 9.6±2.0 (p<0.006), compared to culture of MM cells in fresh media. In contrast, CM from unconditioned MSCs increased growth of HR MM cells by 2.6±0.6 (p<0.01) fold but had minor effect on growth of LR MM cells. CM from MSCs preconditioned with HR MM cells increased growth of LR and HR MM cells by 5.7±0.1 (p<0.0002) and 2.6±1.2 (p<0.04), compared to culture of MM cells in CM from unconditioned MSCs, respectively. Growth of LR MM cells was higher by 2.9±0.3 fold using CM from MSCs preconditioned with HR MM cells than by using CM from MSCs preconditioned with LR MM cells (p<0.005).To determine the role of cell-cell contact, we compared the effect of the preconditioned MSCs and their CM on growth of LR and HR MM cells. Growth of LR MM cells (p< 0.003) and HR MM cells (p< 0.005) was higher when cultured in CM than in co-culture with MSCs. These data imply that soluble factors from preconditioned MSCs support MM cell proliferation and that adhesion of MM cells to MSCs may restrain proliferation.Genes overexpressed in preconditioned MSCs included growth factors (e.g. IL6) and receptors (e.g. EDNRA); genes underexpressed include factors associated with activity of osteoblasts (e.g. ITGBL1) and adipocytes (IGFBP2).A proteomic analysis showed a reduced level of the secreted factors IGFBP2 and ITGBL1 and increase level of IL6 in CM from MSCs preconditioned with HR MM cells compared to CM from unconditioned MSCs. IGFBP2 mediates local bioavailability of IGF1 and IGF2 and is also involved in bone formation and angiogenesis independently of the IGF axis. ITGBL1 is involved in osteoblastogenesis whereas EDNRA is known to be expressed by pericytes.Global gene expression profiles from patient material showed that EDNRA and IGFBP2 are not expressed in MM cells but are highly expressed in cultured MSCs compared to hematopoietic cells in buffy coat BM samples. EDNRA is overexpressed (p<0.005) whereas IGFBP2 is underexpressed (p<0.005) in whole BM biopsy samples from MM patients with HR disease compared to patients with LR disease (p<0.005) and in Focal Lesion compared to random BM biopsies taken from the same patients (p<0.0000006 for EDNRA and p<0.02 for IGFBP2).IHC staining of patients’ bone biopsies showed higher numbers of EDNRA+ mesenchymal-like cells in MM (n=10) than MGUS/AMM (n=10, p<0.0003) and in HR MM BM than LR MM BM (p<0.03). IHC analysis also revealed that IGFBP2 is highly expressed by immature adipocytes and that its expression in these cells is reduced in HR MM BM.Conclusion:Preconditioning of MSCs is essential for promoting growth of MM cells from LR patients. Factors altered in MSCs by HR MM cells are linked to signaling pathways known to directly stimulate MM cell growth and markers associated with distinct MSC lineages changed in HR MM niche. DisclosuresDavies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Barlogie:Signal Genetics: Patents & Royalties. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.