Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart

Cytokines and growth factors secreted by CD105⁺CD34^- cells <i>in vitro</i>.

<p>(A) An exemplary image of a membrane used for the analysis of 80 cytokines and growth factors secreted by CD105⁺CD34^- cells. (B) Densitometric analysis of 80 cytokines and growth factors secreted by CD105⁺CD34^- cells indicates that CD105⁺CD34^- cells secrete mainly IL-6, IL-8, and GRO molecules (n = 5).</p

Human Cardiac Mesenchymal Stromal Cells with CD105⁺CD34^- Phenotype Enhance the Function of Post-Infarction Heart in Mice

<div><p>Aims</p><p>The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105⁺CD34^- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.</p><p>Methods and Results</p><p>MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105⁺CD34^- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.</p><p>Conclusion</p><p>In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105⁺CD34^- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.</p></div

Changes in a size of the scar and fibrosis in the post-infarction heart of a mouse 7 weeks after MI.

<p>(A B C) Representative images illustrating a post-infarction scar in the mouse heart in a (A) Sham group or after administration of (B) PBS^- or (C) CD105⁺CD34^- cells. The images are composed of several shots (magn. 4x). (D) The area of the post-infarction scar is significantly smaller after administration of CD105⁺CD34^- cells compared to the control group, in which PBS^- was administered (n = 14); **p<0.01 by the U Mann–Whitney test. (E F G) Representative images illustrating collagen (pink) deposited between the fibers of the muscle tissue (green) in the mouse heart in a (E) Sham group or after administration of (F) PBS^-, or (G) CD105⁺CD34^- cells. The sections with the greatest ratio of post-infarction scar to the whole stained area were used for examination (magn. 40x). (H) A statistically significant reduction of fibrosis was observed in the border area of the post-infarction scar after administration of CD105⁺CD34^- cells compared to the control group after administration of PBS^- (n = 14); **p<0.01 by the U Mann–Whitney test.</p

Therapeutic potential of CD105⁺CD34^- cells tested in a mouse model of hindlimb ischemia.

<p>(A) CD105⁺CD34^- cells treated mice demonstrated improved functional outcomes compared to the control mice (PBS^-) (n = 5; experiment repeated twice). (B) The number of blood vessels was higher at day 14 in mice after administration of CD105⁺CD34^- cells compared to the control mice after administration of PBS^- (n = 10; 10 muscles per group were analyzed, in each muscle 10 pictures were taken). *p<0.05 #p = 0.056 **p<0.01 by the Mann-Whitney U test.</p

MSC-CM increased the expression of CD206 in BMDM.

<p>The incubation of MSC-CM with BMDM greatly increased the percentage of CD206⁺CD86⁺ and CD206⁺CD86^- macrophages in comparison with control BMDM cells and BMDM cells incubated in medium with LPS; n = 6 *p<0.05; **p<0.01 by one-way analysis of variance (ANOVA), using Tukeys’ multiple comparison test for post hoc analysis for the groups: CD86⁺CD206^- and CD86⁺CD206⁺; by Kruskal–Wallis one-way analysis of variance, using multiple comparison of mean ranks for all groups, for CD86^-CD206⁺ group.</p

MSC with CD105⁺CD34^- phenotype modulate inflammation after administration into infarcted mouse heart.

<p>(A) The number of leukocytes (CD45⁺ cells) infiltrating the post-infarcted heart were significantly lower 1 day after CD105⁺CD34^- cells administration; n = 3. (B) The level of M0 macrophages (% of CD45⁺F480⁺ cells) accumulated in the infarcted heart was higher after CD105⁺CD34^- cells administration; n = 3. (C) CD105⁺CD34^- cells significantly increased the M2/M1 ratio 1 day after the cells administration; n = 3 *p<0.05 compared to the control (PBS^-) group by the Student’s t-test.</p

The presence of M2 macrophages after administration of CD105⁺CD34^- cells into the post-infarction mouse heart.

<p>(A) 1 day after implantation of CD105⁺CD34^- cells (lamin A+C, green) into the border area of the post-infarction scar, small amounts of pro-inflammatory macrophages with the M1 phenotype (iNOS, red) around the transplanted human cells were observed (magn. 20x). (B) 1 day after CD105⁺CD34^- cells (lamin A+C, green) implantation an increase in the number of anti-inflammatory, and proangiogenic macrophages with the M2 phenotype (CD206, red) in their surroundings was observed. The similar phenomenon was observed: (C) (D) 3 days after implantation of CD105⁺CD34^- cells (lamin A+C, green) and (E) (F) 7 days after implantation of CD105⁺CD34^- cells (lamin A+C, green) (magn. 20x).</p

Capillary density within the area of the post-infarction scar 7 weeks after MI.

<p>(A B C) Representative images presenting the number of blood vessels within the area of the post-infarction scar. Magn. 20x. (A) in mice after administration of PBS^- (B) in mice after administration of CD105⁺CD34^- cells. (C) Within the scar, a significant increase in the number of blood vessels in mice after administration of CD105⁺CD34^- cells compared to the control mice after administration of PBS^- was observed. n = 7; ** p<0.01 by the Student’s t-test.</p

Changes in left ventricular ejection fraction (LVEF) 6 weeks after the cells administration.

<p>LVEF increased by 5.75% between treated groups at 49 day. Day 0 –LVEF (baseline); day 7 –LVEF seven days after MI induction, the day of administration of CD105⁺CD34^- cells or PBS^-: day 49 –LVEF on the day of the collection of the hearts. PBS^-: n = 16; CD105⁺CD34^-: n = 18; **p<0.01; *p<0.05; # p = 0.0946. Comparisons between groups and within groups were performed by the one-way analysis of variance (ANOVA) with repeated measures with post-hoc Tukey tests.</p