Cardiac repair in a mouse model of acute myocardial infarction with trophoblast stem cells

Abstract

Various stem cells have been explored for the purpose of cardiac repair. However, any individual stem cell population has not been considered as the ideal source. Recently, trophoblast stem cells (TSCs), a newly described stem cell type, have demonstrated extensive plasticity. The present study evaluated the therapeutic effect of TSCs transplantation for heart regeneration in a mouse model of myocardial infarction (MI) and made a direct comparison with the most commonly used mesenchymal stem cells (MSCs). Transplantation of TSCs and MSCs led to a remarkably improved cardiac function in contrast with the PBS control, but only the TSCs exhibited the potential of differentiation into cardiomyocytes in vivo. In addition, a significantly high proliferation level of both transplanted stem cells and resident cardiomyocytes was observed in the TSCs group. These findings primary revealed the therapeutic potential of TSCs in transplantation therapy for MI.

Figure 1. Characterization of TSCs and MSCs.

(A) TSCs expressed the specific marker, Cdx2, by immunofluorescent staining. (B) TSCs expressed markers of Cdx2, Tfap2c, Fgfr2, and Eomes by RT-PCR, but did not express Oct4 (*P < 0.01). Data are represented as mean ± SD.

Figure 2. Cardiac function and remodeling after cell transplantation.

(A–D) Sequential echocardiographical assessments of the infarcted mice at baseline, 2 weeks, and 3 weeks showed an increase in EF and FS, as well as, a decrease in LVIDd and LVIDs in TSCs- or MSCs- treated animals as compared to the PBS group. (E) Infarct size expressed as a percentage of left ventricular area in the cell therapy groups was significantly smaller than the PBS treated group. (F,G) TIM and WTBZ in cell-treated hearts were substantially higher than the PBS-treated hearts. (H) Quantification of interstitial fibrosis in the border zone of the three groups revealed that the cell therapy decreased the fibrosis of hearts after MI. (I) Quantification of CD31⁺ capillaries suggested that the density of vessels in the cell-treated groups were significantly higher than in the PBS-treated group both in the infarct and border zones. (J) Quantification of Tunel⁺ cells showed that the apoptotic cells in the cell-engrafted groups were significantly lower than in the PBS-treated group at the border zone, but no difference was seen in the infarct zone among the three groups. n = 8 for each group. *P < 0.05, MSCs-treated group vs. PBS-treated group; ^#P < 0.05, TSCs-treated group vs. PBS-treated group. Data are depicted as mean ± SD.

Figure 3. Retention of MSCs and TSCs after transplantation.

(A) The retention of injected cells was detected by the percentage of GFP⁺ in all the cells in every field under fluorescent microscopy, and nuclei were counterstained with DAPI. (B) Quantification of GFP⁺ cells to total cells showed that the retention of TSCs was higher than the MSCs (*P < 0.05) (Table S1). (C) The co-expression of GFP and Tunel indicated the apoptosis of implanted cells in vivo. (D) Quantification of co-expression of Tunel and GFP cells showed no difference between TSCs and MSCs in apoptosis after transplantation (Table S3). (E) The co-expression of GFP and pH3 indicated the proliferation of implanted cells in vivo. (F) Quantification of co-expression of pH3 and GFP cells showed that TSCs exhibited more proliferation than MSCs in vivo (*P < 0.05) (Table S2). n = 6 for each group. Data are depicted as mean ± SD.

Figure 4. Transdifferentiation of TSCs and MSCs in vivo.

(A) Confocal imaging of the TSCs-engrafted hearts and fluorescent immunostaining for α-actinin demonstrated evidence for cardiomyocytes differentiation; nuclei were counterstained with DAPI. (B,C) The sections displayed in B and C showed that MSCs and TSCs gave rise to endothelial-like cells by CD31-staining and participated in the formation of the vasculature as assessed by vWF staining in vivo. The arrows in B identifies co-staining of GFP and CD31. The boxed region in C is shown at higher magnification in the right panel. n = 6 for each group.

Figure 5. Cell proliferation after cell transplantation.

(A) Representative Ki67-stained histological sections at 3-weeks after cell transplantation. (B) Quantification of Ki67⁺ cells showed that proliferating cells in the TSCs-engrafted groups were significantly higher than the MSCs-treated and PBS-treated groups both in the infarct and border zones; however, no difference was observed between the MSCs-treated group and PBS-treated group (n = 8 for each group). (C) Representative pH3-stained sections showed the proliferation of endogenous cardiomyocytes in the border zone at 3 weeks after cell transplantation. The boxed region is shown at higher magnification in the right panel. (D) Quantification of pH3⁺ cells showed that the endogenous proliferation cells in the TSCs-treated group were significantly higher than in the MSCs-treated group (n = 6 for each group) (Table S4). *P < 0.05, TSCs-treated group vs. MSCs-treated group; ^#P < 0.05, TSCs-treated group vs. PBS-treated group. Data are represented as mean ± SD.

Figure 6. Microarray analysis of the hearts after cell therapy.

(A,B) Heat map and hierarchical clustering. The heat map shows the result of the two-way hierarchical clustering of miRNAs and samples. Each row represents a miRNA (fold change >2.0) and each column represents a sample (n = 3 for each group). (A) Showed the different miRNAs between MSCs and PBS groups. (B) Showed the different miRNAs between TSCs and PBS groups. The color scale shown in the top panel illustrates the relative expression level of a miRNA in the certain slide: red color represents a high relative expression level; green color represents a low relative expression level (Table S5). (C,D) Validation of miRNA microarray results by RT-PCR (fold change >2.0 and P < 0.05). (C) showed the confirmed miRNAs between MSCs and PBS groups (n = 3 for each group). The confirmed miRNAs are provided in Table S6. D showed the confirmed miRNAs between TSCs and PBS groups (n = 3 for each group). Data are depicted as mean ± SD.