Source For Cell-based Therapies Research Articles

Generation of self-organized autonomic ganglion organoids from fibroblasts.

Neural organoids have been shown to serve as powerful tools for studying the mechanism of neural development and diseases as well as for screening drugs and developing cell-based therapeutics. Somatic cells have previously been reprogrammed into scattered autonomic ganglion (AG) neurons but not AG organoids. Here we have identified a combination of triple transcription factors (TFs) Ascl1, Phox2a/b, and Hand2 (APH) capable of efficiently reprogramming mouse fibroblasts into self-organized and networked induced AG (iAG) organoids, and characterized them by immunostaining, qRT-PCR, patch-clamping, and scRNA-seq approaches. The iAG neurons exhibit molecular properties, subtype diversity, and electrophysiological characteristics of autonomic neurons. Moreover, they can integrate into the superior cervical ganglia following transplantation and innervate and control the beating rate of co-cultured ventricular myocytes. Thus, iAG organoids may provide a valuable tool to study the pathogenesis of autonomic nervous system diseases and screen for drugs, as well as a source for cell-based therapies.

Insights and future directions for the application of perinatal derivatives in eye diseases: A critical review of preclinical and clinical studies

Perinatal derivatives (PnD) are gaining interest as a source for cell-based therapies. Since the eye is easily accessible to local administration, eye diseases may be excellent candidates to evaluate novel therapeutic approaches. With this work, we performed a systematic review of published preclinical and clinical studies addressing PnD in the treatment of ocular diseases. We have set two specific objectives: (i) to investigate the current level of standardization in applied technical procedures in preclinical studies and (ii) to assess clinical efficacy in clinical trials. Hereto, we selected studies that applied amniotic membrane (hAM) and mesenchymal stromal cells derived from amniotic membrane (hAMSC), placenta (hPMSC), umbilical cord (hUC-MSC) and Wharton's Jelly (hUC-WJ-MSC), excluding those where cells were not transplanted individually, following a systematic PubMed search for preclinical studies and consultation of clinical studies on https://clinicaltrials.gov and https://www.clinicaltrialsregister.eu/. Our bibliographic search retrieved 26 pre-clinical studies and 27 clinical trials. There was a considerable overlap regarding targeted ocular structures. Another common feature is the marked tendency towards (i) locally administered treatments and (ii) the PnD type. In the cornea/ocular surface, hAM was preferred and usually applied directly covering the ocular surface. For neuroretinal disorders, intra-ocular injection of umbilical or placental-derived cells was preferred. In general, basic research reported favourable outcomes. However, due to lack of standardization between different studies, until now there is no clear consensus regarding the fate of administered PnD or their mode of action. This might be accountable for the low index of clinical translation. Regarding clinical trials, only a minority provided results and a considerable proportion is in "unknown status". Nevertheless, from the limited clinical evidence available, hAM proved beneficial in the symptomatic relief of bullous keratopathy, treating dry eye disease and preventing glaucoma drainage device tube exposure. Regarding neuroretinal diseases, application of Wharton's Jelly MSC seems to become a promising future approach. In conclusion, PnD-based therapies seem to be beneficial in the treatment of several ocular diseases. However, much is yet to be done both in the pre-clinical and in the clinical setting before they can be included in the daily ophthalmic practice.

Urethral injection of dedifferentiated fat cells ameliorates sphincter damage and voiding dysfunction in a rat model of persistence stress urinary incontinence

PurposeDedifferentiated fat (DFAT) cells are mature adipocyte-derived multipotent cells that can be applicable to cell-based therapy for stress urinary incontinence (SUI). This study developed a persistence SUI model that allows long-term evaluation using a combination of vaginal distention (VD) and bilateral ovariectomy (OVX) in rats. Then, the therapeutic effects of DFAT cell transplantation in the persistence SUI model was examined.MethodsIn total, 48 Sprague–Dawley rats were divided into four groups and underwent VD (VD group), bilateral OVX (OVX group), VD and bilateral OVX (VD + OVX group), or sham operation (Control group). At 2, 4, and 6 weeks after injury, leak point pressure (LPP) and histological changes of the urethral sphincter were evaluated. Next, 14 rats undergoing VD and bilateral OVX were divided into two groups and administered urethral injection of DFAT cells (DFAT group) or fibroblasts (Fibroblast group). At 6 weeks after the injection, LPP and histology of the urethral sphincter were evaluated.ResultsThe VD + OVX group retained a decrease in LPP with sphincter muscle atrophy at least until 6 weeks after injury. The LPP and urethral sphincter muscle atrophy in the DFAT group recovered better than those in the fibroblast group.ConclusionsThe persistence SUI model was created by a combination of VD and bilateral OVX in rats. Urethral injection of DFAT cells inhibited sphincter muscle atrophy and improved LPP in the persistence SUI model. These findings suggest that the DFAT cells may be an attractive cell source for cell-based therapy to treat SUI.

Dexamethasone priming enhances stemness and immunomodulatory property of tissue-specific human mesenchymal stem cells

BackgroundHuman Mesenchymal Stem Cells (hMSCs) represent a promising cell source for cell-based therapy in autoimmune diseases and other degenerative disorders due to their immunosuppressive, anti-inflammatory and regenerative potentials. Belonging to a glucocorticoid family, Dexamethasone (Dex) is a powerful anti-inflammatory compound that is widely used as therapy in autoimmune disease conditions or allogeneic transplantation. However, minimal immunomodulatory effect of hMSCs may limit their therapeutic uses. Moreover, the effect of glucocorticoids on the immunomodulatory molecules or other regenerative properties of tissue-specific hMSCs remains unknown.MethodHerein, we evaluated the in vitro effect of Dex at various dose concentrations and time intervals, 1000 ng/ml, 2000 ng/ml, 3000 ng/ml and 24 h, 48 h respectively, on the basic characteristics and immunomodulatory properties of Bone marrow derived MSC (BM-MSCs), Adipose tissue derived MSCs (AD-MSCs), Dental Pulp derived MSC (DP-MSCs) and Umbilical cord derived MSCs (UC-MSCs).ResultsThe present study indicated that the concentration of Dex did not ramify the cellular morphology nor showed cytotoxicity as well as conserved the basic characteristics of tissue specific hMSCs including cell proliferation and surface marker profiling. However, quite interestingly it was observed that the stemness markers (Oct-4, Sox-2, Nanog and Klf-4) showed a significant upregulation in DP-MSCs and AD-MSCs followed by UC-MSCs and BM-MSCs. Additionally, immunomodulatory molecules, Prostaglandin E-2 (PGE-2), Indoleamine- 2,3-dioxygenase (IDO) and Human Leukocyte Antigen-G (HLA-G) were seen to be upregulated in a dose-dependent manner. Moreover, there was a differential response of tissue specific hMSCs after pre-conditioning with Dex during mixed lymphocyte reaction, wherein UC-MSCs and DP-MSCs showed enhanced immunosuppression as compared to AD-MSCs and BM-MSCs, thereby proving to be a better candidate for therapeutic applications in immune-related diseases.ConclusionDex preconditioning improved the hMSCs immunomodulatory property and may have reduced the challenge associated with minimal potency and strengthen their therapeutic efficacy.Graphical Preconditioning of tissue specific hMSCs with dexamethasone biomanufacturers the enhanced potential hMSCs with better stemness and immunomodulatory properties for future therapeutics.

Differentiation of Mesenchymal Stem Cells Into Cardiac-like Cells by Co-induction of Lentiviruses Containing Mir-1 and Myocd in Chitosan Collagen Hydrogel Scaffold

Background and Objectives Cardiovascular disease is one of the leading causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources of cell-based therapies in heart regeneration. There are several approaches to differentiate MSCs into cardiac-like cells, such as genetic modification. In addition, using of 3D culture, such as hydrogels, increases the efficiency of differentiation. Methods In the present study, lentiviruses containing microRNA 1 (miR- 1) and myocardium (Myocd) were co-transducted to mouse adipose-derived MSCs. Three days after, transduced MSCs were transferred to a hydrogel containing chitosan and collagen. After 21 days, the differentiation of encapsulated cells was evaluated. In this regard, the expression of cardiac markers such as NK2 homeobox 5 (Nkx2-5), GATA binding protein 4 (Gata4) and troponin T type 2 (Tnnt2) at the level of gene and protein were investigated. Results The results of real-time quantitative polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by transfer to composite hydrogel increased the expression of cardiac markers. Conclusion The use of 3D culture such as chitosan/collagen hydrogel improves the differentiation of MSCs and subsequently obtains more mature cells for use in cell-based regenerative medicine

Human umbilical cord mesenchymal stem cells confer potent immunosuppressive effects in Sjögren’s syndrome by inducing regulatory T cells

Background Primary Sjögren’s syndrome (SS) is a lymphoproliferative disease with a chronic autoimmune disorder characterized by mononuclear cell (MNC) infiltration of notably the lacrimal and salivary glands. As mesenchymal stem cells (MSCs) regulate series of immunological responses partially by regulating proportion of CD4+ T cells and inducing an immunosuppressive local milieu, umbilical cord MSCs (UC-MSCs) are being considered as a novel source for cell-based therapies against primary SS. This study aimed to investigate the feasibility of UC-MSCs in treatment of SS and to explore the possible mechanism(s) with the special emphasis on regulatory T cells (Tregs). Methods Potent immunosuppressive effects of human UC-MSCs on SS were explored in vivo and in vitro. To study the effects of human UC-MSCs on the development and progression of SS, human UC-MSCs were administered before disease onset (preventive protocol) and after disease occurrence (therapeutic protocol) in non-obese diabetic (NOD) mice. In human study, the effect of human UC-MSCs on T cells from SS patients was studied. Results In both protocols, the histopathology of submandibular and sublingual salivary glands showed decreased inflammatory infiltrates. In vitro, human UC-MSCs exhibited potent suppressive effects on responses of MNCs in NOD mice and T cells in SS patients. Such inhibitory effects were coupled with decreased production of proinflammtory cytokines interferon-γ, interleukin (IL)-6, tumor necrosis factor-α and increased production of IL-10 (n = 10, p < .01). The frequency of CD4+Foxp3+T cells in the spleen of NOD recipients was elevated (n = 6, p < .05). Conclusion Human UC-MSCs are capable of inducing CD4+Foxp3+ T cells in both NOD mice and human in vitro. Human UC-MSCs effectively interfere with the autoimmune attack in the course of SS by inducing an in vivo state of T cell unresponsiveness and the upregulation of Tregs.

Fucoidan Rescues p-Cresol-Induced Cellular Senescence in Mesenchymal Stem Cells via FAK-Akt-TWIST Axis.

Mesenchymal stem cells (MSCs) are a source for cell-based therapy. Although MSCs have the potential for tissue regeneration, their therapeutic efficacy is restricted by the uremic toxin, p-cresol, in chronic kidney disease (CKD). To address this issue, we investigated the effect of fucoidan, a marine sulfated polysaccharide, on cellular senescence in MSCs. After p-cresol exposure, MSC senescence was induced, as indicated by an increase in cell size and a decrease in proliferation capacity. Treatment of senescent MSCs with fucoidan significantly reversed this cellular senescence via regulation of SMP30 and p21, and increased proliferation through the regulation of cell cycle-associated proteins (CDK2, CDK4, cyclin D1, and cyclin E). These effects were dependent on FAK-Akt-TWIST signal transduction. In particular, fucoidan promoted the expression of cellular prion protein (PrPC), which resulted in the maintenance of cell expansion capacity in p-cresol-induced senescent MSCs. This protective effect of fucoidan on senescence-mediated inhibition of proliferation was dependent on the TWIST-PrPC axis. In summary, this study shows that fucoidan protects against p-cresol-induced cellular senescence in MSCs through activation of the FAK-Akt-TWIST pathway and suggests that fucoidan could be used in conjunction with functional MSC-based therapies in the treatment of CKD.

Stem cell-based therapies for neurological disorders

Cell-based therapies have been previously performed using fetal tissues for some central nervous system (CNS) disorders, such as Parkinson’s disease. However, it can be difficult to collect a large number of cells for transplantation. Recent studies revealed that some stem cells can act as potential sources of cell-based therapies for degenerative and damaged areas in the CNS. In addition, stem cells can be used as cellular delivery vehicles for brain tumor because of tumor-tropic migratory capacity. Embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and induced pluripotent stem (iPS) cells are the most attractive stem cells. iPS cells can be efficiently differentiated to neural stem cells and have the possibilities to overcome the ethical issues associated with ES cells. Therefore, cell-based therapies using iPS cells can be developed specifically for neurological disorders. In this article, we review the characteristics of ES cells, MSCs, and iPS cells as cell sources for stem cell-based therapies, and then discuss preclinical data and ongoing clinical trials for the CNS disorders.

Abstract 201: High Fat Diet Selectively Decreases the Bone Marrow Lin-/c-kit+ Cell Population in Aging Mice Through Increased ROS Production

Bone marrow-derived c-kit-positive (c-kit + ) cells are a potential source for cell-based therapy and tissue repair and regeneration. The ability of tissue repair and regeneration is significantly decreased in the aging population for various reasons. Western diet usually has increased level of fat. The present study was designed to evaluate the effect of long-term high fat diet on bone marrow c-Kit positive cells and the role of ROS in aging mice. Aging male wild-type (WT) C57BL/6 mice (40 weeks) were fed with high fat diet (HFD) for 3 months with regular diet as the control. To evaluate the role of reactive oxygen species (ROS) in mediating the effect of HFD on bone marrow cell population, a separate group of mice were treated with N-acetylcysteine (NAC) to reduce ROS production. Bone marrow (BM) and blood cells were harvested and prepared in the mice after 3 months of HFD treatment for flowcytometry analysis for Sca-1+, or c-Kit+, or CD133+ cells. The lineage negative (Lin-) and c-Kit positive (Lin-/c-Kit+) cell population was significantly decreased in the BM, not in blood, in the mice with HFD, while no significant change was observed in Sca-1+ or CD133+ cell populations in the BM or blood. The BM Lin-/c-Kit+ cells also exhibited increased intracellular ROS level, increased level of apoptosis, and deceased level of proliferation in HFD-treated mice. NAC treatment significantly decreased intracellular ROS level and normalized the BM Lin-/c-Kit+ cell population in HFD mice. In conclusion, the present study demonstrated that long-term HFD selectively decreased BM Lin-/c-Kit+ cell population in the aging mice through increased ROS production.

Characteristics of human adipose derived stem cells in scleroderma in comparison to sex and age matched normal controls: implications for regenerative medicine

BackgroundAdipose-derived stem cells (ADSCs) are emerging as an alternative stem cell source for cell-based therapies. Recent data suggest that autologous ADSC-enriched micrografting improves the effects of facial involvement in systemic sclerosis (SSc). We have extensively characterised ADSCs from SSc patients and compared their phenotype and function to healthy age- and sex-matched control ADSCs.MethodsADSCs were isolated and characterised from a cohort of six SSc patients (ADSC-SSc) and were compared to six healthy age- and sex-matched controls (ADSC-N). Cell surface phenotype lineage commitment was explored by flow cytometric analysis of mesenchymal and hematopoietic markers and by the capacity to differentiate to chondrogenic, osteogenic, and adipogenic lineages. Functional activities of ADSCs were assessed by biochemical and cellular assays for proliferation, metabolism, adhesion, morphology, migration, and invasion.ResultsUpon characterization of ADSC-SSc, we found that there was no alteration in the phenotype or surface antigen expression compared to healthy matched control ADSCs. We found that the differentiation capacity of ADSC-SSc was equivalent to that of ADSC-N, and that ADSC-SSc did not display any morphological or adhesive abnormalities. We found that the proliferation rate and metabolic activity of ADSC-SSc was reduced (p < 0.01). We found that the migration and invasion capacity of ADSC-SSc was reduced (p < 0.01) compared to healthy matched control ADSCs.ConclusionsThis study provides important findings that can differentially characterise ADSCs from SSc patients. Results indicate that the surface phenotype and differentiation capacity of ADSCs from SSc patients are identical to healthy matched ADSCs. While the findings indicate that the proliferation and migration capacity of ADSC-SSc is reduced, ADSC-SSc are capable of ex-vivo culture and expansion. These findings encourage further investigation into the understanding by which ADSCs can impact upon tissue fibrosis.

Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow.

The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies.

Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.

Clinical application of oligodendrocyte precursor cells for cell-based therapy.

Oligodendrocyte precursor cells (OPCs), which give rise to mature oligodendrocytes (OLs), play important roles in maintaining white matter function. Even during the adulthood period, OPCs comprise roughly 5% of all cells in the forebrain and retain a capability to become myelinated OLs. Recently, OPCs have been proposed as a novel source for cell-based therapy. For the purpose, OPCs can be obtained from embryonic stem cells, induced pluripotent stem cells, and directly converted cells derived from patients. Here, we will provide a brief review of the potential of using OPCs as a cell-based therapy for treating various neurological diseases.

Proof of concept studies exploring the safety and functional activity of human parthenogenetic-derived neural stem cells for the treatment of Parkinson's disease.

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

Potential Cell Source for Cell-Based Therapy and Tissue Engineering Applications: Urine-Derived Stem Cells

Potential Cell Source for Cell-Based Therapy and Tissue Engineering Applications: Urine-Derived Stem Cells

Urine-Derived Stem Cells for Tissue Repair in the Genitourinary System

Tissue-specific stem cells can more efficiently repair the tissue from which they originate, compared to other types of adult stem cells. Human urine-derived stem cells (USCs), a subpopulation of cells constantly exposed to urine, possess biological characteristics of stem cells, and thus are an optimal cell source for tissue repair in the genitourinary system. USCs display clonogenicity, high proliferation capacity, stem cell marker expression profiles, and multipotent differentiation capacity. These cells can more efficiently differentiate into the cell types of the genitourinary tract, particular in urothelial cells, renal podocytes, and endothelial cells, compared to other adult stem cells. In addition, USCs secrete numerous growth factors and cytokines to support cell growth, differentiation, resident stem cell recruitment, and immunomodulatory properties. In vivo studies in rodent models of diabetic erectile dysfunction, urinary tract incontinence, and renal dysfunction demonstrated that USCs significantly enhanced organ function via angiogenesis and transdifferentiation, and showed anti-inflammatory and anti-fibrotic properties after implantation of USCs. USCs can be obtained via a simple, non-invasive, and low-cost approach. Thus, USCs offer an extremely promising cell source for cell-based therapy in androurology.

Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media.

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70–80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

Abstract 17048: Exosomes From Induced Pluripotent Stem Cells Deliver Cardioprotective Mirnas and Prevent Cardiomyocyte Apoptosis in the Ischemic Myocardium

Induced pluripotent stem (iPS) cells exhibit enhanced survival and proliferation in ischemic myocardium and thus represent a promising source of cell-based therapy for myocardial ischemia-reperfusion (MIR) injury. However, the therapeutic application of iPS cells is limited by their tumorigenic potential. We hypothesized that iPS cells can transmit cytoprotective signals to cardiomyocytes via exosomes, nanovesicles that contain proteins, mRNAs and microRNAs (miRNAs) that can regulate cell function. Exosomes secreted from mouse cardiac fibroblast (CF)-derived iPS cells (iPS-exo) were purified from conditioned medium and confirmed by electron micrograph and protein expression of the exosome markers CD63 and Tsg101. We observed that iPS-exo are taken up by cultured H9C2 cardiomyocytes with high efficiency in time-dependent fashion. Uptake of iPS-exo protects H9C2 cells against H2O2-induced oxidative stress by inhibiting caspase 3/7 activation (P&lt;0.05, n=6). To investigate whether iPS-exo treatment can protect against MIR injury, we injected exosomes secreted by CF (CF-exo), iPS-exo, or PBS (control) intramyocardially into mouse ischemic myocardium before reperfusion. After 24hs, TUNEL analysis showed that iPS-exo reduced cardiomyocyte apoptosis by about 66% in comparison with PBS control (p&lt;0.05, n=6), and by about 50% in comparison with CF-iPS (P&lt;0.05, n=6). Western blotting demonstrated that iPS-exo treatment significantly decreased the active caspase3 compared with PBS and CF-exo treatment (P&lt;0.05, n=3). Furthermore, iPS-exo deliver cardioprotective miRNAs, including Nanog-regulated miR-21 and HIF-1α-regulated miR-210, to H9C2 cardiomyocytes in vitro. In conclusion, iPS-exo are biological nanoparticles carrying survival messages that can be efficiently transmitted to cardiomyocytes to protect against MIR.

Mechanisms of Natural Gene Therapy in Dystrophic Epidermolysis Bullosa

Revertant mosaicism has been reported in several inherited diseases, including the genetic skin fragility disorder epidermolysis bullosa (EB). Here, we describe the largest cohort of seven patients with revertant mosaicism and dystrophic EB (DEB), associated with mutations in the COL7A1 gene, and determine the underlying molecular mechanisms. We show that revertant mosaicism occurs both in autosomal dominantly and recessively inherited DEB. We found that null mutations resulting in complete loss of collagen VII and severe disease, as well as missense or splice-site mutations associated with some preserved collagen VII function and a milder phenotype, were corrected by revertant mosaicism. The mutation, subtype, and severity of the disease are thus not decisive for the presence of revertant mosaicism. Although collagen VII is synthesized and secreted by both keratinocytes and fibroblasts, evidence for reversion was only found in keratinocytes. The reversion mechanisms included back mutations/mitotic recombinations in 70% of the cases and second-site mutations affecting splicing in 30%. We conclude that revertant mosaicism is more common than previously assumed in patients with DEB, and our findings will have implications for future therapeutic strategies using the patient's naturally corrected cells as a source for cell-based therapies.

P0024 IL-6 induces malignant transformation of rat mesenchymal stem cells in association with enhanced STAT3 signalling

Background Mesenchymal stem cells (MSCs) have the potential to be a source for cell-based therapies. However, MSCs can undergo malignant transformation in the tumour microenvironment where a high level of IL-6 is present. In this study, we investigated the role of IL-6 and STAT3 signalling in malignant transformation of MSCs. Methods Rat MSCs were isolated and indirectly co-cultured with C6 glioma cells. Co-culture of MSCs with astrocytes was used as a control. After 7 days of culture, the cells were assessed for malignant transformation using MTT immunofluorescence staining. The levels of hepatocyte growth factor, IL-6, and basic fibroblast growth factor, and the expression of STAT3 and soluble IL-6 receptor (sIL-6R), in the cultured cells and the conditioned media were measured using RT-PCR, ELISA, and western blotting. The expression levels of STAT3 downstream targets, cyclin D1 and Bcl-xl, were determined as well. Findings Our data show that almost all of the MSCs became phenotypically malignant after indirect co-culture with glioma cells, which was confirmed by tumour formation assays when these cells were injected into nude mice. The expression of IL-6 was significantly increased in the MSCs co-cultured with glioma cells, which was associated with significantly increased expressions of sIL-6R, GP130, STAT3, phosphorylated STAT3, cyclin D1, and Bcl-xl. Similar results were obtained when the MSCs were treated with IL-6. Furthermore, by treating the co-cultured MSCs with glioma by STA-21 to block the constitutive STAT3 signalling, the risk of MSCs tumour-like transformation in the tumour microenvironment was reduced. Interpretation These data suggest that IL-6 plays a critical role in the malignant transformation of rat MSCs, which is associated with an enhancement of the STAT3 signalling pathway in the tumour microenvironment.