In stem-cell research a major difficulty is caused by the lack of distinctive features that allow the identification of human mesenchymal stem cells (hMSC). Until now, there has been no specific marker and the most common way to identify hMSC is by their characteristic stem-cell properties: self-replication and differentiation potential.However, these findings can only be revealed retrospectively, and, once differentiated, hMSC lose their stem-cell character. The aim of this study was to establish four-colour immunofluorescence of several markers simultaneously in order to address the problem of how to identify hMSC on the single-cell level. The four markers collagen-I, collagen-IV, fibronectin and CD44 are known to be expressed by hMSC. Antibody binding was detected using secondary antibodies conjugated to FITC, Alexa546, TexasRed and AMCA. Because the distinction between Alexa546 and TexasRed was not possible on conventional digital images using standard filter sets, we performed spectral image acquisition. The image was subsequently decomposed into its pure spectral components, which permitted linear unmixing. Using this procedure we were able to demonstrate four-colour immunofluorescence on hMSC. With the possibility of using more sophisticated marker profiles and / or additional markers, four-colour immunofluorescence offers the opportunity of identifying hMSC on the single-cell level without performing differentiation assays.

Renal impairment because of CNI contributes to long-term morbidity. Therefore, CNI avoiding or sparing treatment strategies are important. In this article, we describe the results of a CNI-free treatment protocol with regard to recovery of renal function. Twenty-eight patients with heart transplantation were switched from CNI regimen to everolimus and mycophenolate, when cGFR was <75 mL/min/1.73 m(2). In all patients, CNI was stopped, when everolimus trough levels of 5-8 ng/L were achieved. Serum creatinine and cGFR were determined before and after 6 and 12 months. Median serum creatinine decreased from 1.2 mg/dL (range 0.7-3.7) before everolimus to 1.0 (range 0.6-1.8) and 1.0 (range 0.5-1.9) mg/dL after 6 and 12 months. Median cGFR was 47.81 (range 18.3-72.6) mL/min/1.73 m(2) before everolimus and 63.1 (range 37.8-108.7) mL/min/1.73 m(2) at six months and 64.8 (range 37.7-106.6) mL/min/1.73 m(2) after 12 months. All changes from baseline to six and 12 months were statistically significant (p < 0.05). Adverse events were infections (n = 3) and rejections (n = 3). Therapy was discontinued in four patients. Conversion to CNI-free immunosuppression resulted in significant improvements of renal function within six months of CNI withdrawal. Side effects are common. However, more studies are required to demonstrate the effectiveness in children.

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