Cardiac Migration of Endogenous Mesenchymal Stromal Cells in Patients with Inflammatory Cardiomyopathy

Figure 1

Flow cytometry analysis strategy and quantification of transcardiac gradients of circulating MSC in CMi. (a) Cultivated MSC (bar indicates 200 μm) isolated from bone marrow were stained with directly FITC-conjugated monoclonal antibodies against human CD45 and CD34, and anti-CD11b directly conjugated to AF488 (all from BD). After setting a morphological gate defined by forward/side scatter (FSC and SSC, R1), CD45⁻CD34⁻CD11b⁻ cells (R2) defined as belonging to both R1 and R2 (first row) were stained with CD73-PE, CD106-APC, CD90-APC, CD29-APC, CD105-APC, or CD44-APC (second row). (b) Measurement of circulating MSC in patients with CMi. MNCs were isolated from peripheral blood using a Ficoll gradient. Circulating MSC were analysed by flow cytometry. First, a regional gate was defined to exclude debris and platelets defined by forward/side scatter (FSC and SSC, R1). The events in R1 are then displayed on a CD45CD34CD11b versus SSC dot plot and a second gate (R2) is produced to include the cluster of CD45⁻CD34⁻CD11b⁻ cells. The right margin was fixed for all patients. A total of at least 100,000 events in R1 and R2 were counted. (c) Quantification of the number of circulating MSC sampled from the aortic root and simultaneously from the coronary sinus in patients with cardiac inflammation (box plots are given as median and IQR; whiskers represent 95% CI). The lines between the boxes represent numbers of CD45⁻CD34⁻CD11b⁻/100 MNC sampled from either the aorta or coronary sinus of the same individual.

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