MALDI‐IMS as a Tool to Determine the Myocardial Response to Syndecan‐2‐Selected Mesenchymal Stromal Cell Application in an Experimental Model of Diabetic Cardiomyopathy

Purpose: Mesenchymal stromal cells (MSC) are an attractive tool for treatment of diabetic cardiomyopathy. Syndecan-2/CD362 has been identified as a functional marker for MSC isolation. Imaging mass spectrometry (IMS) allows for the characterization of therapeutic responses in the left ventricle. This study aims to investigate whether IMS can assess the therapeutic effect of CD362+-selected MSC on early onset experimental diabetic cardiomyopathy. Experimental Design: 1 × 106 wild type (WT), CD362−, or CD362+ MSC are intravenously injected into db/db mice. Four weeks later, mice are hemodynamically characterized and subsequently sacrificed for IMS combined with bottom-up mass spectrometry, and isoform and phosphorylation analyses of cardiac titin. Results: Overall alterations of the cardiac proteome signatures, especially titin, are observed in db/db compared to control mice. Interestingly, only CD362+ MSC can overcome the reduced titin intensity distribution and shifts the isoform ratio toward the more compliant N2BA form. In contrast, WT and CD362− MSCs improve all-titin phosphorylation and protein kinase G activity, which is reflected in an improvement in diastolic performance. Conclusions and Clinical Relevance: IMS enables the characterization of differences in titin intensity distribution following MSC application. However, further analysis of titin phosphorylation is needed to allow for the assessment of the therapeutic efficacy of MSC

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362(-), and CD362(+) MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model. Materials: For this purpose, 1 x 10(6) WT, CD362(-), or CD362(+) MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Lepr(db)/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis. Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362(-), and CD362(+) MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362(+) MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362(-) MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3(+) and CD68(+) cells in db/db mice, whereas only splenocytes from CD362(-)- and CD362(+)-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice. Conclusion: CD362(+) MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362(-) MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362(+) MSC application was insufficient to improve diastolic function

Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease

Background: Mesenchymal stromal cells (MSC) possess immunomodulatory properties and low immunogenicity, both crucial properties for their development into an effective cellular immunotherapy. They have shown benefit in clinical trials targeting liver diseases; however the efficacy of MSC therapy will benefit from improvement of the immunomodulatory and immunogenic properties of MSC. Methods: MSC derived from human umbilical cords (ucMSC) were treated for 3 days in vitro with various inflammatory factors, interleukins, vitamins and serum deprivation. Their immunogenicity and immunomodulatory capacity were examined by gene-expression analysis, surface-marker expressions, IDO activity, PGE2 secretion and inhibition of T cell proliferation and IFNγ production. Furthermore, their activation of NK cell cytotoxicity was investigated via CD107a expre

Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion.

BACKGROUND Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool. METHODS Human umbilical cord-derived MSCs (ucMSCs) were cultured for 3 days with interferon (IFN)γ, transforming growth factor (TGF)β or a multi-factor combination (MC; IFNγ, TGFβ and retinoic acid). In addition, ucMSCs were culture expanded for 14 days. Phenotypical changes and T-cell proliferation inhibition capacity were examined. Genome-wide DNA methylation was measured with Infinium MethylationEPIC Beadchip. RESULTS Upon priming, ucMSCs exhibited a different immunophenotype and ucMSC(IFNγ) and ucMSC(MC) had an increased capacity to inhibit T-cell proliferation. DNA methylation patterns were minimally affected by priming, with only one significantly differentially methylated site (DMS) in IFNγ- and MC-primed ucMSCs associated with autophagy activity. In contrast, 14 days after culture expansion, ucMSCs displayed minor phenotypical and functional changes but showed >4000 significantly DMSs, mostly concerning genes involved in membrane composition, cell adhesion and transmembrane signalling. DISCUSSION These data show that DNA methylation of MSCs is only marginally affected by priming, whereas culture expansion and subsequent increased cellular interactions have a large impact on methylation. On account of this study, we suggest that DNA methylation analysis is a useful quality control tool for culture expanded therapeutic MSCs