Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair

Abstract

Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac-derived cells as three-dimensional cardiospheres may recapitulate a stem cell niche-like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self-assembled into stem cell niche-like structures in vitro in suspension culture, while exhibiting greater proportions of c-kit(+) cells and upregulated expression of SOX2 and Nanog. Pathway-focused polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunostaining revealed enhanced expression of stem cell-relevant factors and adhesion/extracellular-matrix molecules (ECM) in cardiospheres including IGF-1, histone deacetylase 2 (HDAC2), Tert, integrin-α(2), laminin-β(1), and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer-cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac-derived cells as cardiospheres mimics stem cell niche properties with enhanced "stemness" and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart.

Figure 1. Growth, proliferation, and recapitulation of stem cell niche-like microenvironment of cells under cardiosphere culture condition

A) Representative images show the growth of cardiac stem cells into cell aggregates and suspended cardiospheres on poly-D-lysine-coated plate (left) or as adherent monolayer cells on a fibronectin-coated plate (right). B) The number of cells increased 2- and 3-fold after 3 and 7 days in monolayer culture, while proliferation was lower in cardiosphere culture. C) Representative images show that two c-kit-positive (green) cardiac stem cells in the central area of a cardiosphere with abundance of collagen IV (red). D) A cluster of c-kit-positive stem cells is localized in the central core of cardiospheres, surrounded by CD105-positive supporting cells. E) Tabular summary of features of cardiac stem cell niches and cardiospheres [NA: not assessed; *based on data presented in Supporting Information Figure 6; ** based on our previous data (Ref #25)].

Figure 2. Expression of c-kit, SOX2, and Nanog in cells under cardiosphere and monolayer culture conditions

A) Compared to the baseline (red line in top histogram), flow cytometry analysis showed that the expression of c-kit in cardiac outgrowth cells was increased after 3 days culture under cardiosphere condition (black line), but decreased under monolayer condition (green line). B) Quantitative data from 6 separate experiments using different human cardiac outgrowth cells. Quantitative qRT-PCR analysis of 4 separate RNA samples from different human cardiac outgrowth cells showed that the expression of SOX2 (C) and Nanog (D) is also significantly higher in cells cultured under cardiosphere than monolayer conditions.

Figure 3. Pathway-focused PCR array and qRT-PCR analyses of the expression of stem cell relevant genes in cells cultured as cardiospheres or monolayers

A) Scatter plot demonstrating 13 out of the 84 stem cell genes up-regulated (red circles) in cells under cardiosphere culture condition. B) The names and fold changes of genes that are up- or down-regulated by greater than 4-fold above baseline in cardiosphere culture compared with monolayer culture. Furthermore, qRT-PCR analysis of 5 separate RNA samples from different human cardiac outgrowth cells confirmed that the expression of IGF-1 (C), TERT (D), and HDAC2 (E) is increased in cardiac stem cells cultured under cardiosphere condition when compared with monolayer condition.

Figure 4. Pathway-focused PCR array analysis of the gene expression of ECM and adhesion molecules in cells cultured as cardiospheres or monolayers

A) The scatter plot demonstrates that 12 of the 84 ECM and adhesion genes are up-regulated (red circles) in cardiospheres relative to monolayer cells. B) The names and fold changes of genes that are up- or down-regulated more than 4-fold in cardiosphere culture compared with monolayer culture. Furthermore, qRT-PCR analysis of 5 separate RNA samples from different human cardiac outgrowth cells confirmed that the mRNA expression of IGTA2 (C), LAMB1 (D), and MMP3 (E) is increased in cells cultured as cardiospheres when compared with monolayer cells.

Figure 5. Engraftment of human cardiac-derived cells 3 weeks after implantation into the infarcted hearts of mice

A) Engraftment of GFP⁺ human cells was infrequently observed in mice receiving the implantation of single cell suspension of CDCs after 3 days culture under monolayer condition (left). However, the survival of GFP⁺ human cardiac-derived cells was more evident in mice receiving a portion of the same initial CDCs after 3 days of culture under cardiosphere condition (2^nd-CSps, right). α-SA: α-sarcomeric actin. B) Quantitative data for cell engraftment (percentage of green (GFP⁺) area/total area) in the infarcted heart after implantation.

Figure 6. Cardiac function after treatment and their relationship to cell engraftment

A) LVEF at baseline and 1 and 3 weeks after treatments. B) Percentage change in LVEF from baseline levels 3 weeks after cell injection. (Control group: saline injection only; Mono group: injection with monolayer-cultured CDCs; 2^nd-CSp group: injection with cardiospheres reformed from twice-passaged CDCs; Pri-CSp group: injection with cardiospheres primarily formed from cardiac-derived cells; Diss-CSp group: injection with cells dissociated from secondary cardiospheres). The percentage of area of engrafted human cardiac-derived cells within the infarcted heart of mice is strongly correlated with either the absolute values of LVEF (C) or the relative LVEF changes to baseline (D), 3 weeks after treatments.

Figure 7. Effect of enzymatic digestion on the expression of ECM and adhesion and the resistance to oxidative stress

A) Western blotting shows that the dissociation of cardiospheres into single cell suspension decreased the expression of integrin-α₂ and laminin-β₁. B) Representative images (upper images) show the TUNEL-positive (red) cardiac-derived cells under cardiosphere and monolayer culture conditions with 24 hrs exposure to 100 μM H₂O₂. Although the number of apoptotic cells (lower bar graph) was lower in cardiospheres than monolayer-cultured cells with 24 hrs exposure to 100 μM H₂O₂, the resistance to oxidative stress in cardiospheres was negated by dissociating the cardiospheres into single cells. (Diss-Mono: dissociated monolayer-cultured cells by 5 minutes trypsin digestion; Mono: twice-passaged CDCs under monolayer culture; Diss-CSp: dissociated cardiospheres by 30 minutes trypsin digestion; CSp: cardiospheres formed from twice-passaged CDCs.).