Human skeletal muscle cells with a slow adhesion rate after isolation and an enhanced stress resistance improve function of ischemic hearts

Abstract

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

Figure 1

Characterization of the RAC and SAC after culture expansion. (a) Each cell population was analyzed for myogenic purity by CD56 flow cytometry. (b) Myogenic gene expression profiles were measured in the RAC and SAC populations. Gene expression values are relative to the endogenous control gene IPO8. (c) SAC populations demonstrated increased expression of all expressed myogenic genes relative to the RAC populations. (d) Detection of other cell types within the RAC and SAC populations: VWF for endothelial cells, CNN1 for smooth muscle cells, GPD1 for adipocytes, DLK1 for preadipocytes (UD, undetectable), PTPRC for blood cells, and FN1 and COL4A1 for fibroblasts. (e) SAC populations displayed decreased expression of FN1 and COL4A1 relative to the RAC populations (*P < 0.05). (f) SAC populations more rapidly differentiated into multi-nucleated myotubes than the RAC populations as signified by increased creatine kinase activity (*P < 0.05). RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 2

Cell proliferation and survival under oxidative and inflammatory stress. (a) Cell proliferation was measured in the RAC and SAC cultures (*P < 0.05). (b,c) SAC demonstrated greater survival under conditions of (b) oxidative and (c) inflammatory stress than the RAC (*P < 0.05). RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 3

Functional assessments. (a) Left ventricular end diastolic area (EDA) was slightly reduced in hearts injected with the RAC and SAC populations when compared with control hearts injected with saline. (b) Hearts injected with SAC displayed stronger left ventricular contractility, as measured by percent fractional area change (FAC), when compared with hearts injected with RAC (*P < 0.05, SAC versus RAC and control; ^†P < 0.05, RAC versus control). RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 4

Infarct histology. (a–c) Representative images were taken from transverse sections of the Masson's trichrome–stained hearts. Muscle tissue is stained red, and collagenous tissue is stained blue. Bars equal 500 µm. (d) SAC-injected hearts demonstrated the greatest reduction of scar tissue when compared with the control (*P < 0.05, SAC versus control). RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 5

Engraftment and proliferation of injected cells in vivo. (a) The arrows designate the engraftment area of the SAC, and the arrowheads indicate host cardiac tissue in Masson's trichrome–stained hearts. Bar equals 125 µm. Image a colocalizes with image b. (b) Fast skeletal myosin heavy chain (MYH)-positive myofibers (green stain, arrows) designate the SAC engraftment region. Cardiac troponin I-positive cardiomyocytes (red, arrowheads) identifies peri-infarct zone. Bar equals 125 µm. (c,d) Mitotic human PCNA-positive cells (red stain) that colocalized with the MYH-positive engraftment region (green stain) were more frequently observed in the SAC-injected hearts when compared with the RAC-injected hearts (*P < 0.05, SAC versus RAC). Cardiac troponin I-positive cardiomyocytes are stained grey, and nuclei are stained blue. PCNA, proliferating cell nuclear antigen; RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 6

Angiogenesis. (a–c) Representative images are shown of CD31 immunostaining. Bars equal 50 µm. (d) The infarcts of hearts transplanted with SAC displayed higher capillary densities when compared with hearts injected with RAC and control vehicle (*P < 0.05, SAC versus RAC and control). RAC, rapidly adhering cell; SAC, slowly adhering cell.

Figure 7

Endogenous cardiomyocyte apoptosis and proliferation. (a) Identification of apoptotic endogenous cardiomyocytes (arrows) by concomitant staining for cardiac troponin I (cTnI, light brown cytoplasmic stain) and TUNEL assay (arrows, dark nuclear stain). Bar equals 50 µm. (b) The number of apoptotic cardiomyocytes was measured in four high power fields (HPF, *P < 0.05, SAC versus control). (c) Proliferating cardiomyocytes (arrow) within the peri-infarct region were identified by colocalization of both Ki-67 (blue nuclear stain) and cTnI (red) within the peri-infarct region. Some of the proliferating cells stained by Ki-67 did not colocalize with cTnI (arrowhead). Bar equals 50 µm. (d) The number of proliferating Ki-67-positive endogenous cardiomyocytes was measured in the peri-infarct region of the hearts (*P < 0.05, SAC versus RAC and control). HPF, high power field; RAC, rapidly adhering cell; SAC, slowly adhering cell, TUNEL, terminal dUPT nick end-labeling.