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. 2020 Dec 16;21(24):9603.
doi: 10.3390/ijms21249603.

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction

Konstantin V Dergilev  1 , Evgeny K Shevchenko  1   2 , Zoya I Tsokolaeva  1   3 , Irina B Beloglazova  1 , Ekaterina S Zubkova  1 , Maria A Boldyreva  1 , Mikhail Yu Menshikov  1 , Elizaveta I Ratner  1 , Dmitry Penkov  1 , Yelena V Parfyonova  1   4
Affiliations

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction

Konstantin V Dergilev et al. Int J Mol Sci. .

Abstract

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24-28%; 0.17-0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.

Keywords: adipose derived mesenchymal stromal cells; cell sheet; heart function; myocardial infarction; stem cell factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Paracrine and expression activity of SCF-MSC. (a) The dynamics of SCF protein level in the conditioned media from SCF-MSC evaluated by immunosorbent assay. The “day 0” bar indicates the basal level of SCF secretion by unmodified MSCs; (b) Expression profile (mRNA level) of SCF-MSC compared to unmodified cells or MSCs that were transduced by green fluorescent protein (GFP)-encoding AAV. The black and white bars indicate MSC-SCF CS and MSC CS groups, respectively. Data are presented as mean ± SD; *—vs. control MSCs, p < 0.05.
Figure 2
Figure 2
Paracrine and expression activity of SCF-MSC. (a) Representative transmission electron micrograph of EVs fractions, scale bar—500 nm; (b) A typical particle size distribution histogram of EVs from rat MSC obtained by Nanoparticle Tracking Analysis (NanoSight LM10); (c) Representative total ion current chromatogram (top) and extracted ion chromatogram (bottom) for SCF peptide (104–124 aa); (d) ESI-MS/MS spectrum of the SCF peptide (104–124 aa).
Figure 3
Figure 3
SCF-MSC CS integration into myocardium tissue and transgene expression at day 5 (a) and 14 (b) after MI induction. Representative images of the infarction area that is covered by the cell sheet comprised of PKH26-labeled cells (red). Specific staining indicates SCF protein (green) distribution throughout the muscle tissue; (c) Bromodeoxyuridine (BRDU) immunohistostaining to identify proliferating cells in the graft and adjacent tissue (day 14). The small square bar indicate the area of the section where the magnified view (large square bar) was taken from; (d) Immunohistostaining for cleaved caspase 3 to identify apoptotic cells in the graft and adjacent tissue (day 14). The small square bar indicate the area of the section where the magnified view (large square bar) was taken from; (e) Immunofluorescent staining for CD105 (green) at day 14; (f) Oil-red staining to identify adipose cells (day 14). Nuclei are stained with DAPI (4′, 6-diamidino-2-phenylindole). Scale bar-100 µm.
Figure 4
Figure 4
Macrophages and mast cells count at day 5 following MI induction and CS transplantation. (a) A representative image of myocardium section stained against CD68 (green), a marker of macrophages/monocytes. Nuclei are stained with DAPI. Scale bar-100 µm. The small square bar indicate the area of the section where the magnified view (large square bar) was taken from; (b) CD68+ cell count in the sham-operated, MSC CS and SCF-MSC CS groups. Data are presented as mean ± SD (Mann–Whitney U-test); (c) A representative image of myocardium section stained with toluidine blue to identify the mast cells. Scale bar-100 µm. The small square bars indicate the area of the section where the magnified view (large square bars) was taken from; (d) Mast cells count in the sham-operated, MSC CS and SCF-MSC CS groups. Data are presented as mean ± SD (Mann–Whitney U-test).
Figure 5
Figure 5
Histological analysis of the ischemic myocardium at day 5 after MI induction and CSs transplantation. (a–c) Representative images of myocardium sections stained for Wt1+ cells (green) from the sham-operated group (a), MSC CS (b) and SCF-MSC CS (c) group. PKH26-labeled MSCs comprising the cell sheet are in red. Nuclei are stained with DAPI. Scale bar-100 µm. The small square bar indicate the area of the section where the magnified view (large square bar) was taken from; (d) Wt+ cells count in the epicardial area of the section; data are presented as mean ± SD (Mann–Whitney U-test), * -SCF-MSC CS vs. control group, p = 0.033. (e–g) Representative images of myocardium sections stained for c-kit+ cells (green) from the sham-operated group (e), MSC CS (f) and SCF-MSC CS (g) group. PKH26-labeled MSCs comprising the cell sheet are in red. Nuclei are stained with DAPI. Scale bar-100 µm. The small square bar indicate the area of the section where the magnified view (large square bar) was taken from; (h) c-kit+ (CD117+) cells count; data are presented as mean ± SD (Mann–Whitney U-test), * -SCF-MSC CS vs. control group, p = 0.0495.
Figure 6
Figure 6
Vascularization of ischemic myocardium at day 5 and 14 after MSC sheet transplantation. (a,b) Representative images of CD31 positive vascular structures (green) in ischemic myocardium covered by CS comprised of pre-labelled MSC (red). Day 14 after MI induction. Nuclei are stained with DAPI (blue). White arrows (b) indicate vascular structures present in the cell sheet. (c) Capillaries and lumen vessel count in FOVs covering the adjacent to CS region. The black asterisk (⁕) indicates statistical significance vs. control group; (d) Capillaries vessel count. The black asterisk (⁕) indicates statistical significance vs. control group; (e) Lumen vessel count. Data are presented as mean ± SD (Mann–Whitney U-test). Scale bar-100 µm.
Figure 7
Figure 7
Morphometric analysis of LV remodeling at day 14 after CS transplantation. (a) Representative images of Mallory trichrome-stained myocardial sections from the sham-operated (control, n = 4), MSC CS (n = 5) and SCF-MSC (n = 6) groups: scar tissue and viable myocardium are in blue and red, respectively. (b) Quantitative analysis of LV morphometric parameters: infarct size, viable myocardium, infarcted wall thickness, LV expansion index. Data are presented as mean ± SD (Mann–Whitney U-test). The black asterisk (⁕) indicates statistical significance; infarct size: MSC SCF CS vs. control group, p = 0.02; viable myocardium: MSC SCF CS vs. control group, p = 0.02; LV expansion: MSC SCF CS vs. control group, p = 0.04; infarcted wall thickness: MSC vs. control group, p = 0.025, MSC SCF vs. control group—not statistically significant.
Figure 8
Figure 8
LV systolic function assessed by TE before and at different time-points (day 7 and 30) after MI and MSC CS (n = 10) or SCF-MSC CS (n = 10) transplantation. The black asterisk (⁕) indicates statistical significance; SCF-MSC CS vs. control group (n = 7), p = 0.037.
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