Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium

Figures

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  Fig. 1. Phenotypic characterization and immunomodulatory effects of rCPCs. Rat CPCs were prepared by sorting for rat CD117 of cells prepared from the right atrial appendage of the heart of Wister Kyoto. A Rat CPCs and rat MSCs characterized for expression of the CD117, CD105, CD90, CD45 and CD31 markers. The expression level of RT1A, RT1D, CD80 and CD86 were measured for rCPCs and compared with rMSCs. B In vitro T cell proliferation in combinations of syngenic, allogenic and xenogenic cells. C–E Results of cytokine analysis by ELISA of syngenic and allogenic and xenogenic combinations. Percentage of T-regulatory cells (T-regs) detected by incubating rCPCs for 5 days with CD4+ T cells isolated from WK and BN spleens in vitro. F, G Show T-reg for syngenic and H, I illustrate T-reg in allogenic combinations. Numerical data are summarized as box and whisker plots with a median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number (n) of rats in each group indicated near the (up/below/on) each respective box and whisker plot

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  Fig. 2. Immunomodulatory effects of rCPCs in vivo. For the in vivo study, one million rCPCs prepared from Wister Kyoto (WK) rat heats were injected intramyocardially immediately after myocardial infarctionin BN rats. Immune cells were isolated five days later. Flow images for detection of T-regs (A) and quantification (B). Flow images for M2 detection (C) and quantification (D). E–H show the percentage of CD8+ and CD4+ cells, respectively. Similarly, I, J show flow images and K, L depict the quantification of CD45R+ and CD3+ respectively. Numerical data are summarized as box and whisker plots with a median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number (n) of rats in each group indicated near the (up/below/on) each respective box and whisker plot

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  Fig. 3. Syngeneic and allogenic, but not xenogeneic CPC transplantation improves cardiac recovery in the rat MI model. For the in vivo study, one million rCPCs prepared from Wister Kyoto (WK) rat heats were injected intramyocardially immediately after MI in Wister Kyoto (syngenic) and Brown Norway (allogenic) rats. The rats were followed for 4 weeks. Evaluation of left ventricular (LV) ejection fraction (A), fractional shortening (B), LV end-systolic volume (C), and cardiac output (D) by echocardiography on day 28. Images (E) and percent scar size (F) from Masson trichrome staining. Myocardial sections were evaluated at 28 days post-transplantation for angiogenesis by co-staining for IB4 and SMA (G–I), and cardiomyocyte proliferation for pHH3/SA (J, K). Hearts were harvested at day 28 post cell transplantation and evaluated for local inflammatory response by histology of stained sections (L) and inflammatory responses were scored in blinded manner (M). Systemic and humoral immune responses were evaluated by measuring circulating anti-donor IgM and IgG in serum (N). Numerical data are summarized as box and whisker plots with a median value (black bar inside box), 25th and 75th percentiles…

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  Fig. 4. Role of GDF15 in cardiac recovery. Basal protein expression of GDF15 in rat CPCs and secretome (A), GDF15 protein expression in rCPCsGDF15KD and rCPCsscramble (B). Cardiac functional parameters were measured after intramyocardial injection of one million rCPCs, rCPCsGDF15KD and placebo separately in the BN rat MI model. C LV ejection fraction, D LV fractional shortening. Representative picture (E) and quantification (F) of Masson trichrome staining in rat hearts. Serum ELISAs for the cytokines IL-10, IL-2, and TNF- α are shown in G, Single-cell suspensions of total heart lysates were used for flow cytometric analysis 5 days after intramyocardial injection of one million rCPCs, rCPCSGDF15KD, or placebo in BN rats. Flow images (H) and quantitative flow results for Tregs (I). Flow images (J) and quantitative flow results (K) for M2 cells. Phosphorylated NF-κB p65 flow images (L) and quantification (M). Whole hearts were also obtained on day 5 for immunohistochemistry studies. Images of phosphorylated NF-κB, FoxP3+, and DAPI staining (N) and quantification (O). Immunohistochemistry of phosphorylated NF-κB and sarcomeric actin with DAPI (P). Numerical data are summarized as box and…

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  Fig. 5. Tregs are essential for myocardial recovery after MI. Cardiac functional parameters were measured after intramyocardial injection of 1 million allogeneic rCPCs, Treg−, Treg+, rCPCs + Treg+ and placebo separately in a nude rat MI model. A and B represent LV ejection fraction and fractional shortening, respectively, at day 1 and day 28. Similarly, single-cell suspensions of total heart lysates were used for flow cytometric analysis of M2 elevation 5 days after intramyocardial injection of 1 million rCPCs, rCPCs + Treg+, or placebo. Representative images (C–E) and quantification (F). Left ventriculat tissue from the heart was used for RNA isolation and quantitative RT-PCR was performed to analyze the expression of G ARG-1, and H IL-10, respectively, from rCPCs, rCPCs + Treg+, and placebo-treated hearts. Numerical data are summarized as box and whisker plots with a median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number (n) of rats in each group indicated near the (up/below/on) each respective box and whisker plot. F and G Immunoblot analysis with…