Mesenchymal Stem Cell Spheroids Research Articles

Solid freeform-fabricated scaffolds designed to carry multicellular mesenchymal stem cell spheroids for cartilage regeneration

Three-dimensional (3D) cellular spheroids have recently emerged as a new trend to replace suspended single cells in modern cell-based therapies because of their greater regeneration capacities in vitro. They may lose the 3D structure during a change of microenvironment, which poses challenges to their translation in vivo. Besides, the conventional microporous scaffolds may have difficulty in accommodating these relatively large spheroids. Here we revealed a novel design of microenvironment for delivering and sustaining the 3D spheroids. Biodegradable scaffolds with macroporosity to accommodate mesenchymal stem cell (MSC) spheroids were made by solid freeform fabrication (SFF) from the solution of poly(D,L-lactide-co-glycolide). Their internal surface was modified with chitosan following air plasma treatment in order to preserve the morphology of the spheroids. It was demonstrated that human MSC spheroids loaded in SFF scaffolds produced a significantly larger amount of cartilage-associated extracellular matrix in vitro and in NOD/SCID mice compared to single cells in the same scaffolds. Implantation of MSC spheroid-loaded scaffolds into the chondral defects of rabbit knees showed superior cartilage regeneration. This study establishes new perspectives in designing the spheroid-sustaining microenvironment within a tissue engineering scaffold for in vivo applications.

Microwell Chips for Selection of Bio‐macromolecules that Increase the Differentiation Capacities of Mesenchymal Stem Cells

Microwell chips (25 mm × 25 mm) are fabricated to select proper substrates for growing three-dimensional (3D) spheroids from mesenchymal stem cells (MSCs). Different bio-macromolecules and their combinations are immobilized on the chip by air plasma treatment and by polyelectrolyte interaction. Only a small number of MSCs (≈10(5) ) are needed for each chip. The expression level of N-cadherin, a cell-adhesion molecule, is used as an indicator for cell-cell interactions. MSC spheroids expressing the highest N-cadherin level also show the greatest osteogenic potential. The microwell chip may be used as an efficient platform to screen bio-macromolecules that enhance the differentiation potential of MSCs.

Substrate-dependent Wnt signaling in MSC differentiation within biomaterial-derived 3D spheroids

A unique biomaterial-based system was developed to generate dynamic three-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs). MSCs were cultured on transparent membranes made of chitosan or those further grafted with hyaluronan (HA) in different densities. MSCs vigorously migrated and were self-assembled into highly mobile 3D spheroids with substrate-dependent upregulation of adhesion molecule N-cadherin. MSC spheroids showed increased expression of Wnt genes/proteins and substrate-dependent cell fate. The correlation of differentiation capacities with Wnt signaling and crosstalk with other pathways such as ERK1/2 or Smad2/3 were observed for MSC spheroids but not for the conventional 2D cultured cells. Wnt3a-mediated canonical Wnt signaling was more active for MSC spheroids derived on chitosan, which were prone to osteogenesis. Wnt5a-mediated non-canonical Wnt signaling was more active for MSC spheroids derived on HA-grafted chitosan, which were prone to chondrogenesis. In particular, the relative importance of Wnt5a-mediated non-canonical vs. Wnt3a-mediated canonical Wnt signals in determining the cell fate was controlled by the grafting density of HA on chitosan. Treatment with the inhibitor of canonical Wnt-associated signaling molecules suppressed the osteogenesis of MSC spheroids on chitosan. This study demonstrates that Wnt signaling of MSCs is distinct in 3D environment and is substrate-dependent. The convenient 3D platform may be used to examine the role of Wnt signaling in controlling MSC fate under different extracellular environments, and potentially applied to study stem cell behavior in regenerative medicine, normal development, and cancer.

Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds

Adipose-derived mesenchymal stem cells (adMSCs) exhibit a high angiogenic activity. Accordingly, their incorporation into tissue constructs represents a promising vascularization strategy in tissue engineering. In the present study, we analyzed whether the efficacy of this approach can be improved by seeding adMSCs as three-dimensional spheroids onto porous scaffolds. Green fluorescent protein (GFP)-positive adMSCs expressing CD13, CD73, CD90 and CD117 were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of spheroids using the liquid overlay technique. Porous polyurethane scaffolds were seeded with these spheroids or a comparable number of individual adMSCs and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice. The vascularization of the implants was analyzed and compared to non-seeded scaffolds by means of intravital fluorescence microscopy and immunohistochemistry. The adMSC spheroids exhibited a homogeneous diameter of ∼270μm and could easily be incorporated into the scaffolds by dynamic seeding. After implantation, they induced a strong angiogenic host tissue response, resulting in an improved scaffold vascularization with a significantly higher functional microvessel density when compared to non-seeded scaffolds and scaffolds seeded with individual adMSCs. Immunohistochemical analyses revealed that a high fraction of ∼40% of all microvessels within the center of spheroid-seeded scaffolds developed from GFP-positive adMSCs. These vessels inosculated with ingrowing GFP-negative vessels of the host. This indicates that adMSC spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside implanted tissue constructs. Thus, adMSC spheroids may be used to increase the efficacy of MSC-based vascularization strategies in future tissue engineering applications.

The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes

Mesenchymal stem cells (MSCs) were recently found to form three-dimensional (3D) multicellular spheroids on chitosan membranes. The exact mechanism of spheroid formation, however, remains unclear. In this study, the regulation of spheroid formation for adipose derived adult stem cells (ADAS) grown on chitosan membranes was examined. By varying the membrane thickness, calcium concentration in culture medium, and acetylation extent of chitosan, the physico-chemical characteristics of chitosan that modulated spheroid formation was elucidated. The capacity of cardiomyogenic differentiation was further evaluated. Results suggested that the calcium binding capacity of chitosan may affect the cell–substrate and cell–cell interactions and critically influence the dynamics of spheroid formation. The intracellular calcium level was elevated for ADAS spheroids on chitosan. Chitosan-bound calcium was observed to enter the cells. The expression of N-cadherin was upregulated for ADAS spheroids on chitosan, evidenced by quantitative RT-PCR and Western blot. After the induction by 5-aza, the expression levels of cardiac marker genes (Gata4, Nkx2.5, Tnnt2, and Myh6) were remarkably enhanced (about four-fold) for ADAS on chitosan vs. tissue culture polystyrene or polyvinyl alcohol. Immunofluorescence staining confirmed the expression of cardiac-associated tight junction protein ZO-1 for ADAS grown on chitosan membranes. The gene expression of Wnt11 was significantly upregulated for ADAS spheroids on chitosan at 3 days and 12 days. We suggested that Wnt11 may be involved in the spheroid formation and cardiomyogenic differentiation of MSCs on chitosan membranes. Spheroids formed on the acetylated chitosan or polyvinyl alcohol membranes failed to show such behavior. The properties of MSC spheroids were therefore determined by the culture substrate.

Stiffening of human mesenchymal stem cell spheroid microenvironments induced by incorporation of gelatin microparticles

Culturing multipotent adult mesenchymal stem cells as 3D aggregates augments their differentiation potential and paracrine activity. One caveat of stem cell spheroids, though, can be the limited diffusional transport barriers posed by the inherent 3D structure of the multicellular aggregates. In order to circumvent such limitations, polymeric microparticles have been incorporated into stem cell aggregates as a means to locally control the biochemical and physical properties of the 3D microenvironment. However, the introduction of biomaterials to the 3D stem cell microenvironment could alter the mechanical forces sensed by cells within aggregates, which in turn could impact various cell behaviors and overall spheroid mechanics. Therefore, the objective of this study was to determine the acute effects of biomaterial incorporation within mesenchymal stem cell spheroids on aggregate structure and mechanical properties. The results of this study demonstrate that although gelatin microparticle incorporation results in similar multi-cellular organization within human mesenchymal stem cell spheroids, the introduction of gelatin materials significantly impacts spheroid mechanical properties. The marked differences in spheroid mechanics induced by microparticle incorporation may hold major implications for in vitro directed differentiation strategies and offer a novel route to engineer the mechanical properties of tissue constructs ex vivo.

Differentiation of human mesenchymal stem cell spheroids under microgravity conditions.

To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or "mesenspheres", of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.

Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes

Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell–substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes ( Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.

The Generation of Three-dimensional Tissue Structures with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow more detailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.

Mesenchymal stem cells promote mammosphere formation and decrease E-cadherin in normal and malignant breast cells.

IntroductionNormal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.ObjectiveWe tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.ResultsWe found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.ConclusionsMSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.

Hierarchically Assembled Mesenchymal Stem Cell Spheroids Using Biomimicking Nanofilaments and Microstructured Scaffolds for Vascularized Adipose Tissue Engineering

AbstractComposite multicellular spheroids composed of mesenchymal stem cells (MSCs) and synthetic biodegradable nanofilaments are fabricated. Extracellular‐matrix‐mimicking nanofilaments, prepared from transverse fragmentation of semicrystalline poly(L‐lactic acid) nanofibers and subsequent surface modification with cell adhesive peptides, are used to form composite multicellular spheroids with MSCs by cellular self‐assembly. The size of the composite spheroids could be readily controlled with the integrated amount of the nanofilaments. The composite spheroids show enhanced adipogenic potential compared to homotypic spheroids. The resultant spheroids are used as building blocks for 3D biohybrid construction with the assistance of a microstructured scaffold fabricated by a direct polymer melt deposition process. An angiogenic growth factor, basic fibroblast growth factor, is also locally delivered in a sustained fashion from the heparinized scaffold surface for facile neovascularization of adipogenic tissue. The produced multiscaled and multifunctional hybrid MSC construct enable the successful formation of vascularized adipose tissue in vivo.

Induction of Intervertebral Disc–Like Cells From Adult Mesenchymal Stem Cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.