Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential.

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.

Expression of blood group genes by mesenchymal stem cells.

Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate- and protein-based membrane structures, defined by blood group antigens, we investigated human bone marrow-derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase-1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2(dim+) H(+) MSCs retain a better 'stemness'. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.

Advanced intercross line mapping suggests that ncf1 (ean6) regulates severity in an animal model of guillain-barre syndrome.

We here present the first genetic fine mapping of experimental autoimmune neuritis (EAN), the animal model of Guillain-Barré syndrome, in a rat advanced intercross line. We identified and refined a total of five quantitative trait loci on rat chromosomes 4, 10, and 12 (RNO4, RNO10, RNO12), showing linkage to splenic IFN-gamma secretion and disease severity. All quantitative trait loci were shared with other models of complex inflammatory diseases. The quantitative trait locus showing strongest linkage to clinical disease was Ean6 and spans 4.3 Mb on RNO12, harboring the neutrophil cytosolic factor 1 (Ncf1) among other genes. Polymorphisms in Ncf1, a member of the NADPH oxidase complex, have been associated with disease regulation in experimental arthritis and encephalomyelitis. We therefore tested the Ncf1 pathway by treating rats with a NADPH oxidase complex activator and ameliorated EAN compared the oil-treated control group. By proving the therapeutic effect of stimulating the NADPH oxidase complex, our data strongly suggest the first identification of a gene regulating peripheral nervous system inflammation. Taken together with previous reports, our findings suggest a general role of Ncf1 and oxidative burst in pathogenesis of experimental autoimmune animal models.

Intranasal delivery of cells to the brain.

The safety and efficacy of cell-based therapies for neurodegenerative diseases depends on the mode of cell administration. We hypothesized that intranasally administered cells could bypass the blood-brain barrier by migrating from the nasal mucosa through the cribriform plate along the olfactory neural pathway into the brain and cerebrospinal fluid (CSF). This would minimize or eliminate the distribution of cellular grafts to peripheral organs and will help to dispense with neurosurgical cell implantation. Here we demonstrate transnasal delivery of cells to the brain following intranasal application of fluorescently labeled rat mesenchymal stem cells (MSC) or human glioma cells to naive mice and rats. After cells crossed the cribriform plate, two migration routes were identified: (1) migration into the olfactory bulb and to other parts of the brain; (2) entry into the CSF with movement along the surface of the cortex followed by entrance into the brain parenchyma. The delivery of cells was enhanced by hyaluronidase treatment applied intranasally 30 min prior to the application of cells. Intranasal delivery provides a new non-invasive method for cell delivery to the CNS.

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability.

BACKGROUND AIMS: Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS: The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS: Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS: The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.

The use of clinically approved small particles of iron oxide (SPIO) for labeling of mesenchymal stem cells aggravates clinical symptoms in experimental autoimmune encephalomyelitis and influences their in vivo distribution.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Mesenchymal stem cells (MSC) have been shown to ameliorate symptoms in experimental autoimmune encephalomyelitis (EAE), a model of MS. Using cloned MSC labeled with clinically approved small particles of iron oxide (SPIO) for treatment of EAE we analyzed the tissue localization of transferred cells. Treatment with unlabeled MSC led to disease amelioration compared to controls. In contrast, treatment with SPIO-labeled MSC lead to increase in disease severity. Treatment with SPIO alone did not alter disease course. After transplantation labeled and nonlabeled MSC were detected in the CNS and the liver with significantly more SPIO-labeled cells present in the CNS. Iron deposition was present in the group treated with SPIO-labeled MSC, indicating that in vivo the initially cell surface-bound iron detached from the MSC. These results could be of great importance for imaging of patients in the clinical setting, indicating that in vivo application of SPIO-labeled MSC needs to be performed with caution because the cell-derived exposure of iron can lead to disease aggravation.

Dual inhibition of proteasomal and lysosomal proteolysis ameliorates autoimmune central nervous system inflammation.

Multiple sclerosis (MS) is a detrimental disease of the central nervous system (CNS) leading to long-term disability. In the course of animal models of multiple sclerosis (experimental autoimmune encephalomyelitis), we find enhanced activity of proteasome subunits beta1i, beta2, beta2i and beta5 in the CNS. We demonstrate that pharmacological inhibition of the proteasome by bortezomib ameliorates experimental autoimmune encephalomyelitis in mice and rats in prophylactic and therapeutic treatment with reduced numbers of T-cells secreting proinflammatory cytokines. The anti-inflammatory effect of proteasome inhibition was accompanied by reduced NF-kappaB activity in the CNS and lymphoid organs. The combined inhibition of proteasomes and lysosomal proteases involved in major histocompatibility complex II antigen presentation further improved therapeutic efficacy. We suggest proteasome inhibition alone or in combination with inhibition of lysosomal proteases as a novel therapeutic strategy against inflammation-induced neurodegeneration in the CNS. We demonstrate the impact of the proteasome and lysosomal proteases on development of autoimmunity.