Human Cardiac-Mesenchymal Stem Cell-Like Cells, a Novel Cell Population with Therapeutic Potential

Abstract

Cardiac stem/progenitors are being used in the clinic to treat patients with a range of cardiac pathologies. However, improvements in heart function following treatment have been reported to be variable, with some showing no response. This discrepancy in response remains unresolved. Mesenchymal stem cells (MSCs) have been highlighted as a regenerative tool as these cells display both immunomodulatory and proregenerative activities. The purpose of this study was to derive a cardiac MSC population to provide an alternative/support to current therapies. We derived human cardiac-mesenchymal stem cell-like cells (CMSCLC), so named as they share some MSC characteristics. However, CMSCLC lack the MSC trilineage differentiation capacity, being capable of only rare adipogenic differentiation and demonstrating low/no osteogenic or chondrogenic potential, a phenotype that may have advantages following transplantation. Furthermore, CMSCLC expressed low levels of p16, high levels of MHCI, and low levels of MHCII. A lack of senescent cells would also be advantageous for cells to be used therapeutically, as would the ability to modulate the immune response. Crucially, CMSCLC display a transcriptional profile that includes genes associated with cardioprotective/cardiobeneficial effects. CMSCLC are also secretory and multipotent, giving rise to cardiomyocytes and endothelial cells. Our findings support CMSCLC as a novel cell population suitable for use for transplantation.

Keywords: cardiac stem cells; cardiosphere-cardiac-derived cells; single cell.

FIG. 1.

Morphology of different primary cells in culture. Representative brightfield images of cells in vitro at passage 3. (A) BM-derived MSCs, (B–D) show images from three different patient-derived cultures of CMSCLC. Brightfield image showing morphology of human CDCs (E) Brightfield image of CMSCLC CFU (F). All scale bars = 100 μm. CMSCLC cultures derived from three different patient tissue samples were assessed for their ability to form CFUs under standard MSC culture conditions, and BM was also assessed for CFU formation ability (G). The number of CFUs formed per culture at passage 1 is represented graphically. MSCs, mesenchymal stem cells; CMSCLC, cardiac-derived mesenchymal stem cell-like cells; BM, bone marrow. Color images are available online.

FIG. 2.

Immunophenotyping of CS-CDCs and CMSCLC. Representative images of FACS histograms showing results of immunophenotyping of CDCs (n = 3) and CMSCLC (n = 3) using cell surface antigens of both hematopoietic lineage-committed cells (CD45 and CD19) and those known to be expressed by MSCs (CD44, CD105, CD166). Controls are isotype control and unstained cells for both cell populations. All cell cultures were immunophenotyped at passage 3. CS-CDCs, cardiosphere-cardiac-derived cells; FACS, flow cytometry.

FIG. 3.

FACS analysis for expression of MHCs and c-kit, and IHC analysis for CD34 expression, by CMSCLC. (A) Representative images of FACS histograms showing expression levels of MHC I and MHC II by CMSCLC. (B) Table showing percentage of cells expressing MHCI and MHCII in three different patient-derived CMSCLC cultures. (C) Representative image of FACS histogram showing expression level of c-kit in CMSCLC and table showing percentage of cells expressing c-kit in three different patient-derived CMSCLC cultures. (D) Cells from the same patient-derived CMSCLC line were cultured for 9 days under normal CMSCLC culture conditions (Control) and under endothelial cell (EC) culture conditions (EC differentiation media) and were analyzed for the expression of CD34. Note lack of CD34 in cells cultured under normal CMSCLC culture conditions (magnification ×400). Scale bars = 100 μm. All cultures were examined at passage 3. IHC, immunohistochemistry.

FIG. 4.

Characterization of trilineage differentiation potential of CDCs and CMSCLC at passage 3. Representative brightfield images, from three different patient tissue samples of (A) CDC and (B) CMSCLC after 28 days in osteogenic culture conditions and before alizarin red staining, demonstrate cellular proliferation and matrix deposition. Images of cells from the same samples but after being stained with alizarin red staining reveal mineralization of osteoid matrix within osteogenic cultures differentiated from CDCs (C), whereas (D) there is no mineralization of culture matrix when CMSCLC were differentiated. Images of CDCs (E) and CMSCLC (F) after 21 days under adipogenic culture conditions stained with oil red O. Note the presence of red stained cells in both cultures. Images of cryosections of cell aggregates formed under prochondrogenic culture conditions after 14 days and after staining with safranin O (counterstained with hematoxylin and fast green) for CDCs (G) and CMSCLC (H). Note that while cell aggregates formed for both CDCs and CMSCLC, chondrogenesis did not occur as evidenced by the absence of safranin O staining for sulfated glycosaminoglycans. Histological evaluation of cell aggregates shows lack of extracellular matrix deposition between cells and hence weak tissue structure. All scale bars = 100 μm. BM-MSCs are shown after culture using the same differentiation protocols as for CDCs and CMSCLC and stained with alizarin red (I), oil red O (J), and safranin O (K). Note BM-MSCs can differentiate to all three lineages. Scale bars for (I, J) = 100 μm. Scale bar for (K) = 400 μm. BM-MSCs, bone marrow-derived MSCs. Color images are available online.

FIG. 5.

Quantification of CMSCLC p16 immunostaining. Representative image of p16 ICC showing results of staining of CMSCLC from patients, 1179, p16 (green), all nuclei stained with DAPI (blue), scale bar = 100 μm (A). CMSCLC cultures derived from three different patient tissue samples were immunostained for the cell senescence marker p16 and counterstained with DAPI. The percentage of p16-positive cells within each culture was calculated by counting five independent fields within each culture. Box-plot analysis (SPSS) showed no significant difference in p16 expression between n = 3 patient cultures (B). Color images are available online.

FIG. 6.

Characterization of cardiac differentiation potential of CDCs and CMSCLC. Representative images of CDC (n = 3 donors) and CMSCLC (n = 4) donors at 14 days under cardiac differentiation culture conditions show that some of both CDC (A) and (B) CMSCLC differentiated cells have a more mature cardiac morphology and express troponin C (green-Alexa Fluor 488). While other cells from the same patient samples cultured under the same conditions but stained for expression of NKX2.5 have a more primitive rounded morphology and express nuclear NKX2.5 (NKX2.5 green-Alexa Fluor 488) in differentiated CDC (C) and differentiated CMSCLC culture (D). White arrows in (C, D) indicate cells shown in inserts of NKX2.5 staining without the DAPI overlay to shown the nuclear localization of NKX2.5. (E–H) Representative images of CMSCLC (n = 4 donors) differentiated for 3 weeks and analyzed by confocal microscopy. (E) Maximum intensity projections of differentiated CMSCLC expressing troponin C (green-Alexa Fluor 488). (Ei) Higher magnification image demonstrates early striations, indicated by yellow arrow. (F) Maximum intensity volume projection of differentiated CMSCLC labeled with cardiac actin (red-Alexa Fluor 594). (G) Maximum intensity volume projection of differentiated CMSCLC labeled with alpha-tropomyosin (red-Alexa Fluor 594). (H, Hi) Maximum intensity volume projection demonstrates striated pattern of alpha-tropomyosin expression, indicated by yellow arrows. For all images, nuclei labeled with DAPI-blue. Scale bars were either 20 or 50 μm as indicated. (I) Bar chart showing percentage of cells expressing NXK2.5 or troponin C, n = 3 for both CDC and CMSCLC cultures analyzed. Color images are available online.

FIG. 7.

Graphical representation of single-cell data representing percentage of CMSCLC expressing genes of interest for n = 3 donors. The percentage of cell expressing each gene is shown. Color images are available online.