We found that NRCVCs cultured in 3D aggregates comprising the same cell population as parallel 2D cultures exhibit subtle but physiologically important alterations in phenotype. Cells cultured in 3D self-assembled into a tissue-like conformation with a superficial layer of ECs resembling the luminal epithelium of native heart tissue. Three-dimensional culture was associated with altered gene expression profiles relative to 2D, and 3D culture resulted in the differential activation of EC migratory pathways. Three-dimensional cultures exhibited decreased ANP and CARP protein levels indicative of improved tissue maturation, and 3D aggregates responded more rapidly to T3 exposure than parallel 2D cultures, consistent with a mature, native-tissue phenotype. These data indicate that 3D culture in the serum-free medium results in the reorganization and phenotypic alteration of the component cells into structures and behaviors similar to those seen in vivo.
Three-dimensional culture systems have significantly advanced our understanding of fundamental relationships between tissue-level organization and component cell function,13,14
and substantial progress has been made in the use of 3D methods to investigate the formation of cardiac tissue.1–12
The phenotypic adaptations of cells when organized in engineered tissues, however, have not been well elucidated. To study aspects of ex vivo
cardiogenesis, we dissociated functional tissue and obtained a mixed cell population representing the original tissue's composition but lacking its multi-cellular organization. It is noteworthy that this cell population is the target population for stem cell and other cell procurement strategies in cardiovascular regenerative therapies and that understanding the controlled assembly of tissue level structures by this population may be critical to the ultimate success of cardiac tissue engineering efforts. In the present study, the formation of 3D aggregates is not entirely surprising as other investigators have used similar low-shear suspension culture models to generate 3D aggregates. The presented work, however, is distinguished by the use of a serum-free medium and highly controlled culture conditions, which severely restrict cell overgrowth ( and ) and which allow the identification of phenotypic differences between component cells in parallel 3D and 2D models.
The up-regulation of EC migratory pathways () and the appearance of a luminal endothelial sheet in the 3D cultures () were striking and unexpected given the propensity of CMs to adhere to surfaces later than other cells (). One possibility is that fluid shear in our 3D suspension-culture system activated the CMs or ECs, which are known to be sensitive to mechanical stimulation.50–54
The level of fluid shear determined for systems like the one employed here, however, is low (<<
and the shear level may not be sufficient to trigger a cellular response. In addition, fluid shear is continuously present throughout the duration of 3D culture, but the Bmp-2
, and Thbs1
expression levels increase and then decrease in both systems (). Finally, Bmp-2
expression, which may drive EC migration, is reportedly unaffected by shear stress in other experiments using similar cells,57
and preliminary studies in our lab to induce Bmp-2
expression in 2D cultures by low level shear exposure have been unsuccessful (data not shown). Thus, although a role for fluid shear cannot be completely ruled out in EC redistribution, it is unlikely that shear contributes to differences in Bmp-2
expression. A second possibility is that differences in Bmp-2 RNA level and EC migration result from coupled interactions between CMs and ECs activated by the 3D contacts permitted in the suspension culture system. BMP-2 is a potential mediator of coordinated EC and CM structure formation in vivo
and CM function is suspected of driving aspects of cardiac morphogenesis during normal development via BMP.58
This is an attractive alternative, but further experiments with 3D models are needed to clarify potential multi-cellular interactions and the mechanisms driving multi-cellular structure formation in vitro
Interestingly, researchers using NRCVC reaggregate culture systems based on serum-containing media do not report exteriorized ECs,7–12
and we initially suspected that the absence of serum in our system may have resulted in a reversal of relative adhesiveness such that CMs adhered first forming a core of cells onto which ECs subsequently adhered. Experiments shown in , however, indicate that CMs adhere later than other cells in our serum-free medium; thus, the appearance of a superficial epithelial layer is not easily accounted for by early CM adhesion to TCPS. Our observations suggest that NRCVCs possess a latent ability to organize an EC sheet after culture initiation and that this ability is accessed in 3D/bioreactor cultures but not in 2D/plate cultures when a serum-free medium is used.
Genomic expression analysis using Affymetrix Chips indicated that a number of genes were differentially expressed in 3D versus 2D culture and that these clustered into six groups (). Evaluation of either the entire 167 genes or the genes in each of the six clusters separately was carried out using the online database for annotation, visualization, and integrated discovery (DAVID) informatics resource from the National Institutes of Health.59,60
After controlling for false discovery using Benjamini-Hochberg corrections, DAVID analysis revealed no thematic relationships among the differentially expressed genes beyond ontologies associated with muscle contraction and muscle development (not shown). Nonetheless, there were physiologically significant differences in gene expression associated with 3D culture, and several of the genes identified in the genomic screening, including Bmp-2
(Cluster 2; also in ), as well as Nppa
(Clusters 1 and 6, respectively; also in ), were analyzed elsewhere in this article. Determining the functional significance of the other expression differences will require additional molecular and proteomic anlayses.
The observed differences in ANP and CARP levels suggest the acquisition of a different and possibly more mature cell phenotype in 3D versus 2D culture. ANP expression from the Nppa
gene has been well studied. ANP is expressed in immature ventricular CMs, but expression decreases over time. In the mature heart, ANP expression is largely restricted to atrial CMs except under pathologic conditions associated with increased ventricular CM stretch (i.e., preload) and hypertrophy.61,62
Since there is no preloading of CMs in our system and no evidence for hypertrophy (), the decrease in ANP is suggestive of CM maturation in the 3D cultures. Similarly, differences in CARP expression from the Ankrd1
gene, which is developmentally down-regulated but reexpressed in hypertrophic ventricular tissue,44,63
suggest that 3D culture encourages CM maturation. As with Bmp-2
, a possible explanation for these differences lies in alteration of cell–cell contacts in 3D. Interestingly, recent work on signaling mechanisms involving Notch pathways, which are triggered by specific cell–cell interactions, has shown that both ANP64,65
levels are controlled by the Notch signaling intermediate Hey-2. Thus, differences in ANP and CARP may be associated with altered cell–cell interactions and the acquisition of a layered, tissue-like geometry in 3D.
Ventricular CMs respond to changes in T3
levels by altering the ratio of α- and β-MyHC.48In vivo
, this isotype switch occurs rapidly when T3
is given to hypothyroid animals.49
Developmentally, the complement of α- and β-MyHC in rodent cardiac ventricles shifts from ~50% α at birth to nearly 100% α by 3 weeks postnatal.67
Thus, the rapid and nearly complete conversion of MyHC mRNA to the α form when 3D cultures were given 3
() is consistent with a shift in hormone sensitivity and the adoption of a more mature CM phenotype.
Taken together, our data indicate that the growth of cardiac ventricular cells in 3D resulted in subtle but significant alterations in cell organization and function. In particular, 3D culture was associated with differences in gene expression, cell migratory signaling, tissue maturation, and responsiveness to hormonal stimulation; these are all differences that impact directly on the design of functional, 3D cardiac tissue. The mechanisms driving alterations in cell function require further investigation but likely involve specific cellular interactions in the 3D environment. This concept is supported by our studies, which emphasized 3D cell masses and did not employ 3D scaffolds. Consideration of the cell interactions that drive the functional alteration of cells and tissues may impact heavily on scaffold design and strategies for tissue formation and may prove critical as the field approaches clinical applications for complex 3D tissues.