Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.
Multipotential stromal cells or mesenchymal stem cells (MSCs), originally isolated as single cell suspensions of bone marrow colonies of fibroblast-like cells adhering to plastic, carry multilineage differentiation potentials
Traditionally, two-dimensional (2D) adherent culture conditions have been used as a standard technique for
2D cell culture is an easy and traditional culture condition; however, it is a highly artificial and less physiological environment, as some
In the regular cell culture condition, anchorage dependent cells, including MSCs, in suspension will fall on the plastic surface by gravity and establish the cell adhesion to the plastic (strictly speaking, to the extracellular matrix (ECM) molecules such as fibronectin adsorbed on the plastic surface of cell culture plates and dishes via cell surface integrins) [
Later methods have improved upon the spinner flask and liquid overlay techniques to generate a more homogeneous population of spheroids. 96-well plates are now commercially available with low attachment surfaces for single spheroid production per well (e.g., 96 Well Ultra-Low Attachment Spheroid Plate from Corning in Corning, NY, or 3D-culture NanoCulture plate from Scivax in Tokyo, Japan); thus, spheroid size is determined by the number of cells in each well [
The formation process of multicellular spheroidal aggregates in low attachment conditions starts with the initial loose cell aggregate formation through integrin-ECM binding followed by the spheroid compaction through enhanced cell to cell connection via homophilic cadherin binding [
Spheroidal cell culture has been used extensively in the field of oncology [
Spheroidal cell culture with pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), is specifically called embryoid body [
Differentiation capability and potential of stem and progenitor cells are generally enhanced in the 3D culture setting. For example, salivary gland-derived progenitor cells can differentiate into hepatocytic and pancreatic islet cell lineages, but these differentiations only take place when the cells are cultured in 3D cell aggregates, not in 2D monolayer [
There are some possible limitations known in the 3D spheroid culture technique. Because of the spheroidal structure, diffusion of nutrients, oxygen, and waste through the interior of the spheroids is compromised in a size-dependent manner [
MSCs cultured in spheroids are spherical inside and elongated outside with an overall reduction of cytoskeletal molecules and ECM. The size of MSCs in spheroids is drastically smaller than cells in 2D monolayer, resulting in 75% reduction in individual cell volume [
Another major difference between 2D monolayer culture and 3D spheroid culture is Young’s elasticity modulus of the materials surrounding the cells, which should also affect cell differentiation [
Microarray analysis showed a drastic change in the gene expression profile in the MSC spheroid culture when compared with MSCs in 2D monolayer culture with upregulation of 1,731 genes and downregulation of 1,387 genes [
Quantitative reverse transcription- (qRT-) PCR is the major method for quantitative analysis of targeted gene expression in current cell biology research. As discussed earlier, gene expression of cytoskeletal molecules including
As discussed earlier, tumor spheroids are an
MSC-based therapeutics is a promising approach in the field of autoimmune diseases, regenerative medicine, and tissue engineering. However, the beneficial effects of MSC-based therapeutics in initial small scale clinical studies are often not substantiated by large randomized-controlled clinical trials, strongly indicating the urgent need of further optimization of cell-based therapy [
Clinical significance of MSC spheroids. Formation of spheroidal aggregates (1) enhances paracrine secretion of angiogenic, antitumorigenic, and pro- and anti-inflammatory factors, (2) improves cell survival, (3) increases differentiation potentials, and (4) delays replicative senescence of MSCs (ANG: angiogenin; ANGPT2: angiopoietin 2; CCL2: chemokine (C-C motif) ligand 2; CCL7: chemokine (C-C motif) ligand 7; FGF2: fibroblast growth factor 2; HGF: hepatocyte growth factor; IL1A: interleukin 1
MSCs exert strong anti-inflammatory or immunomodulatory effects and MSC-based therapeutics is regarded as promising approaches against immune-mediated diseases, such as graft versus host disease. Multiple molecules, such as indoleamine dioxygenase-1 (IDO1) or prostaglandin E2 (PGE2), have been identified to mediate MSCs’ strong anti-inflammatory effects [
Interestingly, strong upregulation of these mediators in MSC spheroids is observed when MSC spheroids are cultured in the regular cell culture medium (i.e., alpha MEM supplemented with FBS), but this upregulation is largely abolished if the MSC spheroids are cultured in the serum and animal component-free chemically defined medium (MesenCult-XF Medium, STEMCELL Technologies, Vancouver, Canada) [
As seen above, spheroidal formation upregulates proinflammatory cytokines (such as IL1A, IL1B, and IL8) and chemokines (such as chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-C motif) ligand 7 (CCL7)) that recruit inflammatory cells, indicating possible proinflammatory properties of MSCs [
Tissue repair and regeneration are an essential biological function for humans. In this complex biological process, numerous types of cells and bioactive mediators are regulated in a temporary and spatially sophisticated manner. The normal repair process of adult tissues, represented with skin in this case, takes place in three phases: inflammation, new tissue formation, and remodeling.
Gene expression of various growth factors and cytokines, including angiogenin (
HGF-mediated antifibrotic effects have been reported for MSCs [
A key feature of MSCs is their multilineage differentiation potentials, which have drawn attention in the field of regenerative medicine [
Another interesting feature of MSC spheroids is that spheroidal formation prolongs replicative lifespan or delays cell senescence of MSCs
Enhanced multilineage differentiation potentials, delayed cell senescent processes, and upregulation of pluripotency marker genes are indicative of enhanced stemness in MSC spheroids. This concept is supported by colony formation assays, which measure the proportion of early progenitors in culture [
Although ease of
IL24 is a multifunctional cancer killing cytokine [
One of the factors limiting the efficacies of MSC therapeutics is posttransplant cell survival [
Despite the promising potential that MSC spheroids have in regenerative medicine and autoimmune diseases, there is limited research on the underlying molecular mechanisms and signaling pathways which initiate and mediate these drastic differences in the gene expression profile and phenotype of MSC spheroids.
Oxygen reaches the inside of spheroids through diffusion, which makes the internal core of spheroids hypoxic [
The self-aggregation process of MSCs initiates caspase-dependent IL1 autocrine signaling. We have previously shown that one of the signaling molecules upregulated by IL1 is early growth response gene-2 (EGR2), a zinc finger transcription factor that regulates PGE2 levels through regulation of
The IL1 autocrine signaling subsequently upregulates chemokine receptors, such as CXCR4, or immunomodulatory mediators, such as TNFAIP6/TSG6, IL6, and PGE2 [
Even though a glimpse of key signaling pathways has been revealed, more studies of crucial molecular events and signaling need to be conducted. For example, the signaling pathways connecting the initial self-aggregation process and the apoptotic process are still unknown. Moreover, the upstream signaling events causing such a drastic change in the gene expression profiles in MSC spheroids [
Epigenetics is defined as an inheritable change in gene expression through DNA methylation, noncoding RNAs, and histone modification, without altering the DNA sequence itself [
MSCs have shown promise in the field of regenerative medicine and 3D MSC culture further enhances such characteristics. Microarray analysis has shown a drastic change in the gene expression profile between monolayer and spheroid cultured MSCs; however, a critical lack of understanding exists with relation to the molecular signaling mediating the enhanced MSC spheroid properties or the improved cell survival. More mechanistic work is definitely needed at the molecular level to better understand and optimize MSC spheroids for clinical applications.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by University of Pittsburgh Schools of Health Sciences (Bridge Funding Category One) and University of Pittsburgh Medical Center Health System (Competitive Medical Research Fund) (Kenichi Tamama).