Delivery Of Mesenchymal Stromal Cells Research Articles

Abstract 196: Genetically Modified Human Mesenchymal Stromal Cells (MSCs) Help Improve Glucose Homeostasis by Reducing Inflammation and Promotes Browning of Visceral Fat

Background: MSCs are multipotent cells that can home-in to a site of inflammation. Upregulation of specific antioxidants in MSCs reduces intracellular inflammation and ROS formation in a hyperglycemic condition. Hypothesis: Antioxidant over-expressed MSCs will reach fat depots, reduce local & systemic inflammation and improve glucose homeostasis in diet-induced obese (DIO, 60% and 45% fat diet) hyperglycemic mouse models. Methods: We used GFP-containing adenoviral constructs to upregulate intracellular (SOD1, SOD2, Catalase) and extracellular (SOD3) antioxidants in human adipose-derived MSCs. The modified MSCs were delivered in DIO mice. Results: In-vitro, SOD2 (mitochondrial anti-oxidant) upregulation showed reduced inflammatory markers IL6 and TNFa mRNA while PCG1A mRNA (a gene upstream of UCP1), upregulated. SOD2 upregulated MSC delivery in both DIO models demonstrated improved glucose tolerance test (GTT) at week 4 compared to SOD1-MSC and Null-MSC (control). Catalase-MSC delivery not only improved GTT but also improved insulin tolerance test (ITT) in 60% DIO mice. Interestingly, RT-PCR of pericardial fat showed significant increases in mRNA expression of both UCP1 (25-100,000-fold) and PRDM-16 (2-10-fold) in both DIO mice models that received antioxidants upregulated MSCs, compared to mice receiving Null MSCs. For omental fat, an increase in mRNA expression of UCP1 was observed in 60% fat DIO mice (1,000-6000 fold) for SODs 1-3 and catalase, while for 45% DIO mice only those receiving SOD1 & SOD2 upregulated MSCs presented UCP1 mRNA upregulation (1,000 to 11,000-fold). Omental Fat histology showed less hyperplastic fat with SOD2 and Catalase-MSCs. UCP1 staining of omental fat was also positive with SOD2-MSC. Inflammatory molecules such as IL-6 and TNF alpha levels by ELISA, were reduced with SOD2-MSC in 60% DIO mice model. Conclusion: We conclude that delivery of antioxidant upregulated MSCs to the inflamed adipocyte depots in diabetic DIO models appear to upregulate UCP1 and PRDM-16 in visceral fat while reducing systemic inflammatory markers, which may explain improvements noted in GTT and ITT. Delivery of modified MSC is a novel & robust therapeutic tool that improves glucose homeostasis in diet induced diabetes.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

Mesenchymal stromal cell therapy in ischemic stroke

Mesenchymal stromal cell therapy in ischemic stroke Ye Zhang, Hong Deng, Chao Pan, Yang Hu, Qian Wu, Na Liu, Zhouping Tang Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People&rsquo;s Republic of China Abstract: Stroke is a clinical disease with high incidence, high disability rate, and high mortality. But effective and safe therapy for stroke remains limited. Adult mesenchymal stromal cells (MSCs) perform a variety of therapeutic functions. MSC delivery improves neurological outcomes in ischemic stroke models via neurorestorative and neuroprotective effects such as angiogenic effects, promoting endogenous proliferation, and reducing apoptosis and inflammation. MSC secretome also showed powerful therapeutic effects as a cell-based therapy in animal experiments. Several clinical trials on MSC implantation via different routes have now been completed in patients with stroke. Although challenges such as immunogenicity of allo-MSCs and large-scale production strategies need to be overcome, MSCs can be considered as a promising potential therapy for ischemic stroke. Keywords: mesenchymal stromal cell, stroke, therapy, transplantation, exosomes

Impact of local injection of brain-derived neurotrophic factor–expressing mesenchymal stromal cells (MSCs) combined with intravenous MSC delivery in a canine model of chronic spinal cord injury

Background aimsThe microenvironment of the chronically injured spinal cord does not allow for axonal regeneration due to glial scarring. To ameliorate this, several therapeutic strategies have been used. We investigated whether combined transplantation of chondroitinase ABC (chABC) and mesenchymal stromal cells (MSCs) genetically modified to secrete brain-derived neurotrophic factor (BDNF) with intravenous (IV) administration of MSCs can promote recovery of hindlimb function after chronic spinal cord injury (SCI). MethodsCanine BDNF-expressing MSCs were generated using a lentivirus packaging protocol. Twelve beagle dogs with experimentally induced chronic SCI were divided into chABC/MSC-green fluorescent protein (GFP), chABC/MSC-BDNF and chABC/MSC-BDNF/IV groups. The MSCs (1 × 107 cells) and chABC were transplanted 3 weeks after SCI in all groups, and IV injection of MSC-GFP (1 × 107 cells) was performed 1 and 2 weeks after MSC transplantation in the chABC/MSC-BDNF/IV group. Spinal cords were harvested 8 weeks after transplantation. ResultsThe dogs in the chABC/MSC-BDNF included groups had significantly improved functional recovery 8 weeks after transplantation compared with those in the chABC/MSC-GFP group. The animals in the chABC/MSC-BDNF/IV group showed significant improvements in functional recovery at 6, 7 and 8 weeks compared with those in the chABC/MSC-BDNF group. Fibrotic changes were significantly decreased in the chABC/MSC-BDNF/IV group. We also observed significant decreases in the expression levels of tumor necrosis factor-α, interleukin-6, COX-2, glial fibrillary acidic protein and GalC and increased expression levels of BDNF, β3-tubulin neurofilament medium, and nestin in the chABC/MSC-BDNF/IV group. ConclusionsWe suggest that transplantation of combined chABC and BDNF-expressing MSCs, along with IV injection of MSCs, is the optimal therapy for chronic SCI.

A Novel Model for Acute Peripheral Nerve Injury in the Horse and Evaluation of the Effect of Mesenchymal Stromal Cells Applied In Situ on Nerve Regeneration: A Preliminary Study

Transplantation of mesenchymal stromal cells (MSCs) to sites of experimentally created nerve injury in laboratory animals has shown promising results in restoring nerve function. This approach for nerve regeneration has not been reported in horses. In this study, we first evaluated the in vitro ability of equine bone marrow-derived MSCs (EBM-MSCs) to trans-differentiate into Schwann-like cells and subsequently tested the MSCs in vivo for their potential to regenerate a transected nerve after implantation. The EBM-MSCs from three equine donors were differentiated into SCLs for 7 days, in vitro, in the presence of specialized differentiation medium and evaluated for morphological characteristics, by using confocal microscopy, and for protein characteristics, by using selected Schwann cell markers (GFAP and S100b). The EBM-MSCs were then implanted into the fascia surrounding the ramus communicans of one fore limb of three healthy horses after a portion of this nerve was excised. The excised portion of the nerve was examined histologically at the time of transection, and stumps of the nerve were examined histologically at day 45 after transplantation. The EBM-MSCs from all donors demonstrated morphological and protein characteristics of those of Schwann cells 7 days after differentiation. Nerves implanted with EBM-MSCs after nerve transection did not show evidence of nerve regeneration at day 45. Examination of peripheral nerves collected 45 days after injury and stem cell treatment revealed no histological differences between nerves treated with MSCs and those treated with isotonic saline solution (controls). The optimal delivery of MSCs and the model suitable to study the efficacy of MSCs in nerve regeneration should be investigated.

In Vivo Bioluminescence Imaging of Transplanted Mesenchymal Stromal Cells and Their Rejection Mediated by Intrahepatic NK Cells.

Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant. Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells. We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R 2 = 0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death. Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.

Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study.

Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL), storage solutions (complete growth medium, Dulbecco's phosphate-buffered saline, and normal saline), storage time in suspension (0–9 h), and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs) and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh) cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%). A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

Mesenchymal Stromal Cells as Cell-Based Therapeutics for Wound Healing.

Chronic wounds are a source of substantial morbidity for patients and are a major financial burden for the healthcare system. There are no current therapies that reliably improve nonhealing wounds or reverse pathological scarring. Mesenchymal stromal cells (MSCs) are a promising source of novel cell-based therapies due to the ease of their harvest and their integral role in the native wound repair process. Recent work has addressed the problems of loss of plasticity and off-target delivery through use of modern bioengineering techniques. Here we describe the applications of MSCs harvested from different sources to the wound healing process and recent advances in delivery of MSCs to targeted sites of injury.

Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model?

Mesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage. The purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma. An orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically "clean" amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a "dirty" amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch's, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0.05 considered significant. Mice receiving intravenous MSCs had a faster time to first detection of pulmonary metastasis (2.93 ± 1.90 days) compared with mice with local injection of MSCs (6.94 ± 6.78 days) or no MSCs (5.93 ± 4.55 days) (p = 0.022). MSC treatment did not influence whether mice developed local recurrence (p = 0.749) or size of recurrent tumors (p = 0.221). MSCs delivered to the surgical site did not promote local recurrence or size of recurrent tumors, but intravenous injection of MSCs did hasten onset of detection of pulmonary metastatic disease. Although local administration of MSCs into a surgical site does not appear to promote either pulmonary metastatic disease or local recurrence, large variation within groups and small numbers diminished statistical power such that a Type II error cannot be ruled out. If MSCs are to be used to augment bone healing in the postlimb salvage setting in patients with osteosarcoma, it will be important to understand their influence, if any, on pulmonary micrometastsis or residual microscopic local disease. Although murine models do not completely recapitulate the clinical scenario, these results suggest that intravenous delivery of MSCs may promote micrometastatic pulmonary disease. Local administration into a surgical wound, even in the presence of residual microscopic disease, may be safe, at least in this murine model, but further investigation is warranted before considering the use of MSCs for clinical use in patients with osteosarcoma.

Failure of intravenous or intracardiac delivery of mesenchymal stromal cells to improve outcomes after focal traumatic brain injury in the female rat.

Mesenchymal stromal cells secrete a variety of anti-inflammatory factors and may provide a regenerative medicine option for the treatment of traumatic brain injury. The present study investigates the efficacy of multiple intravenous or intracardiac administrations of rat mesenchymal stromal cells or human mesenchymal stromal cells in female rats after controlled cortical impact by in vivo MRI, neurobehavior, and histopathology evaluation. Neither intravenous nor intracardiac administration of mesenchymal stromal cells derived from either rats or humans improved MRI measures of lesion volume or neurobehavioral outcome compared to saline treatment. Few mesenchymal stromal cells (<0.0005% of injected dose) were found within 3 days of last dosage at the site of injury after either delivery route, with no mesenchymal stromal cells being detectable in brain at 30 or 56 days post-injury. These findings suggest that non-autologous mesenchymal stromal cells therapy via intravenous or intracardiac administration is not a promising treatment after focal contusion traumatic brain injury in this female rodent model.

Paracrine action of mesenchymal stromal cells delivered by microspheres contributes to cutaneous wound healing and prevents scar formation in mice

Background aimsAccumulating evidence suggests that mesenchymal stromal cells (MSCs) participate in wound healing to favor tissue regeneration and inhibit fibrotic tissue formation. However, the evidence of MSCs to suppress cutaneous scar is extremely rare, and the mechanism remains unidentified. This study aimed to demonstrate whether MSCs—as the result of their paracrine actions on damaged tissues—would accelerate wound healing and prevent cutaneous fibrosis. MethodsFor efficient delivery of MSCs to skin wounds, microspheres were used to maintain MSC potency. Whether MSCs can accelerate wound healing and alleviate cutaneous fibrosis through paracrine action was investigated with the use of a Transwell co-culture system in vitro and a murine model in vivo. ResultsMSCs cultured on gelatin microspheres fully retained their cell surface marker expression profile, proliferation, differentiation and paracrine potential. Co-cultures of MSCs and fibroblasts indicated that the benefits of MSCs on suppressing fibroblast proliferation and its fibrotic behavior induced by inflammatory cytokines probably were caused by paracrine actions. Importantly, microspheres successfully delivered MSCs into wound margins and significantly accelerated wound healing and concomitantly reduced the fibrotic activities of cells within the wounds and excessive accumulation of extracellular matrix as well as the transforming growth factor-β1/transforming growth factor-β3 ratio. ConclusionsThis study provides insight into what we believe to be a previously undescribed, multifaceted role of MSC-released protein in reducing cutaneous fibrotic formation. Paracrine action of MSCs delivered by microspheres may thus qualify as a promising strategy to enhance tissue repair and to prevent excessive fibrosis during cutaneous wound healing.

Role of ECM/peptide coatings on SDF-1α triggered mesenchymal stromal cell migration from microcarriers for cell therapy

Many cell therapies rely on the ability of mesenchymal stromal cells (MSCs) to diffuse and localize throughout the target tissue – such as tumoral and ischemic tissues-, in response to specific cytokine signals, rather than being concentrated at the site of implantation. Therefore, it is fundamental to engineer biomaterial carriers as reservoirs, from which cells can migrate, possibly in a controlled manner. In this work, microcarriers (μCs) made of polylactic acid are characterized as MSC delivery vehicles capable of modulating key chemotactic pathways. The effect of different functionalization strategies on MSC migratory behavior from the μCs is studied in vitro in relation to SDF-1α/CXCR4 axis, – a major actor in MSC recruitment, chemotaxis and homing. Collagen and arginine–glycine–aspartic acid (RGD) peptides were either covalently grafted or physisorbed on μC surface. While stable covalent modifications promoted better cell adhesion and higher proliferation compared to physisorption, the functionalization method of the μCs also affected the cells migratory behavior in response to SDF-1α (CXCL12) stimulation. Less stable coatings (physisorbed) showed sensibly higher number of migrating cells than covalent collagen/RGD coatings. The combination of physic-chemical cues provided by protein/peptide functionalization and stimuli induced by 3D culture on μCs improved MSC expression of CXCR4, and exerted a control over cell migration, a condition suitable to promote cell homing after transplantation in vivo. These are key findings to highlight the impact of surface modification approaches on chemokine-triggered cell release, and allow designing biomaterials for efficient and controlled cell delivery to damaged tissues.

Intrathecal delivery of mesenchymal stromal cells protects the structure of altered perineuronal nets in SOD1 rats and amends the course of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder resulting in a lethal outcome. We studied changes in ventral horn perineuronal nets (PNNs) of superoxide dismutase 1 (SOD1) rats during the normal disease course and after the intrathecal application (5 × 105 cells) of human bone marrow mesenchymal stromal cells (MSCs) postsymptom manifestation. We found that MSCs ameliorated disease progression, significantly improved motor activity, and prolonged survival. For the first time, we report that SOD1 rats have an abnormal disorganized PNN structure around the spinal motoneurons and give different expression profiles of chondroitin sulfate proteoglycans (CSPGs), such as versican, aggrecan, and phosphacan, but not link protein-1. Additionally, SOD1 rats had different profiles for CSPG gene expression (Versican, Hapln1, Neurocan, and Tenascin-R), whereas Aggrecan and Brevican profiles remained unchanged. The application of MSCs preserved PNN structure, accompanied by better survival of motorneurons. We measured the concentration of cytokines (IL-1α, MCP-1, TNF-α, GM-CSF, IL-4, and IFN-γ) in the rats' cerebrospinal fluid and found significantly higher concentrations of IL-1α and MCP-1. Our results show that PNN and cytokine homeostasis are altered in the SOD1 rat model of ALS. These changes could potentially serve as biological markers for the diagnosis, assessment of treatment efficacy, and prognosis of ALS. We also show that the administration of human MSCs is a safe procedure that delays the loss of motor function and increases the overall survival of symptomatic ALS animals, by remodeling the recipients' pattern of gene expression and having neuroprotective and immunomodulatory effects. Stem Cells 2014;32:3163–3172

Non-enzymatic dissociation of human mesenchymal stromal cells improves chemokine-dependent migration and maintains immunosuppressive function

Background aimsHuman bone marrow–derived mesenchymal stromal cells (MSC) can suppress inflammation; therefore their therapeutic potential is being explored in clinical trials. Poor engraftment of infused MSC limits their therapeutic utility; this may be caused by MSC processing before infusion, in particular the method of their detachment from culture. MethodsEnzymatic methods of detaching MSC (Accutase and TrypLE) were compared with non-enzymatic methods (Cell Dissociation Buffer [CDB], ethylenediamine tetra-acetic acid and scraping) for their effect on MSC viability, chemokine receptor expression, multi-potency, immunomodulation and chemokine-dependent migration. ResultsTrypLE detachment preserved MSC viability and tri-lineage potential compared with non-enzymatic methods; however, this resulted in near complete loss of surface chemokine receptor expression. Of the non-enzymatic methods, CDB detachment preserved the highest viability while retaining significant tri-lineage differentiation potential. Once re-plated, CDB-detached MSC regained their original morphology and reached confluence, unlike with the use of other non-enzymatic methods. Viability was significantly reduced with the use of ethylenediamine tetra-acetic acid and further reduced with the use of cell scraping. Addition of 1% serum during CDB detachment led to higher MSC numbers entering autophagy and increased MSC recovery after re-plating. TrypLE and CDB-detached MSC suppressed CD3+CD4+CD25− T-cell proliferation, although TrypLE-detached MSC exhibited superior suppression at 1:20 ratio. CDB detachment retained surface chemokine receptor expression and consequently increased migration to CCL22, CXCL12 and CCL4, in contrast with TrypLE-detached MSC. ConclusionsThis study demonstrates that non-enzymatic detachment of MSC with the use of CDB minimizes the negative impact on cell viability, multipotency and immunomodulation while retaining chemokine-dependent migration, which may be of importance in MSC delivery and engraftment in sites of injury.

Effects Of a Novel Ceramic Biomaterial On Immune Modulatory Properties and Differentiation Potential Of Mesenchymal Stromal Cells

Bone is one of the most frequently transplanted tissues, with allografts and autografts accounting for more than 80% of total grafts. Synthetic biomaterials in association with autologous or allogeneic mesenchymal stromal cells (MSCs) represent a valid alternative in orthopaedic and maxillofacial surgery. Aim of REBORNE consortium is to perform clinical trials using standardized protocols based on advanced biomaterials and MSCs. Aim of our Unit was to assess MSC immune modulatory properties in presence of a novel biomaterial consisting of hydroxyapatite and tricalcium-phosphate (HA/TCP, Biomatlante, France) as scaffold for MSC delivery. We assessed immune modulatory properties, in terms of immunophenotype, inhibitory and anti-apoptotic effects, of MSCs of different origin when associated with the HA/TCP biomaterial, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), adipose-derived MSCs (ASCs) and cord blood-derived MSCs (CB-MSCs). The culture of all MSCs with HA/TCP, did not modulate the anti-apoptotic and suppressive features of all MSCs toward sorted-T, B and NK lymphocytes as compared to standard culture setting. When exposed to inflammatory cytokines, such as IFN-γ and TNF-α, all MSCs were induced to acquire the suppressive phenotype, characterized by MHC-1 and CAM molecules up-regulation, and de novo expression of HLA-DR molecules.The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype, as indicated by the up-regulation of osterix and osteocalcin transcription. The differentiation process is promoted in all MSC types, but especially in BM-MSCs, by dexamethasone and, to a higher extent, BMP-4 treatment. In view of using MSCs for advanced therapies in allogeneic setting, we evaluated the immunogenicity and immune modulatory features of pre-differentiated MSCs in association with HA/TCP towards both innate and adaptive immune cells, as well as the molecular pathways involved in MSC-mediated immune regulation. MSC-derived osteoblasts did not induce immune cell activation, as demonstrated by co-culture of unstimulated immune effector cells with pre-differentiated MSCs. We found a lower suppressive capability of pre-differentiated MSCs towards stimulated T and NK cells. However, some inhibitory effects could be exerted by BMP-4 treated-MSCs, and could be related to the presence of undifferentiated MSCs in culture, which could inhibit lymphocyte proliferation. The balance between the number of bone depositing osteocyte-like MSCs and immunosuppressive undifferentiated MSCs could be useful, in vivo, to promote bone healing and to reduce local inflammation, respectively. We then investigated in pre-differentiated MSCs the molecular mechanisms involved in the inhibition of T cell proliferation, by interfering with IDO and COX-2 activation. We found that pre-differentiated MSCs switched on their suppressive machinery by activating both IDO, which plays a central role in immune regulation, and COX-2, whose role is not significant in undifferentiated MSCs. COX-2 is produced rapidly as inflammation mediator after fracture, and recent data highlighted the role of COX-2 in improving fracture healing.In conclusion, this study increases the knowledge on MSC biology and gives pre-clinical data concerning the use of allogeneic MSC in combination with ceramic scaffolds to treat bone defects. Disclosures:No relevant conflicts of interest to declare.

Are cultured mesenchymal stromal cells an option for immunomodulation in transplantation?

Are cultured mesenchymal stromal cells an option for immunomodulation in transplantation?

Rationale and design of the first randomized, double-blind, placebo-controlled trial of intramyocardial injection of autologous bone-marrow derived Mesenchymal Stromal Cells in chronic ischemic Heart Failure (MSC-HF Trial)

Stem cell therapy is an emerging treatment modality in cardiovascular disease. The best cell type and delivery method in different cardiovascular diseases remain to be determined. The MSC-HF trial is a phase 2, single-center, double-blind, randomized, placebo-controlled trial of intramyocardial delivery of autologous bone-marrow derived mesenchymal stromal cells (MSCs) in patients with chronic ischemic heart failure. A total of 60 patients will be randomized in a 2:1 pattern to receive intramyocardial injections of either MSCs or placebo. Patients will be followed up for 12 months. Bone marrow will be obtained by aspiration from the iliac crest. Mesenchymal stromal cells will be isolated, and culture will be expanded for 6 to 8 weeks. A total of 12 to 15 MSC or placebo injections will be placed in an ischemic viable region of the myocardium using the electromechanical NOGA-XP system (Biologics Delivery Systems Group, Johnson & Johnson, Irwindale, CA). The primary endpoint is change in left ventricle end-systolic volume, measured by magnetic resonance imaging (MRI) or computed tomography (CT) at 6-month follow-up. Secondary endpoints are left ventricle ejection fraction, ventricular volumes, wall thickness, and systolic wall thickening measured by MRI or CT in addition to measurement of myocardial scar tissue by MRI. Other secondary endpoints are safety of treatment, clinical symptoms and functional capacity, weekly angina attacks, use of short-term nitroglycerine, and quality of life. A randomized, double-blind, placebo-controlled, clinical trial of intramyocardial delivery of MSCs in patients with ischemic heart failure has been set up to confirm the positive findings in open-labeled clinical trials.

176 Mesenchymal Stromal Cell Therapy for Bronchiolitis Obliterans Syndrome – Preliminary Data in Humans

Bone marrow derived mesenchymal stromal cells (MSC) represent an attractive cellular therapeutic target given their immunosuppressive properties, ease of isolation and propagation and relative immunologic privilege. Preclinical studies and human trials in graft versus host disease suggest potential efficacy in bronchiolitis obliterans syndrome (BOS). The purpose of this dual centre, single dose, phase 1 study (www.clinicaltrials.gov NCT01175655) was to explore the feasibility and safety of delivery of allogeneic MSC in patients with BOS complicating lung transplantation.

Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation

Abstract 1924Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries.Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante).Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results:We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC.Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix.These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures:No relevant conflicts of interest to declare.