Coculture Pellets Research Articles

Co-culture pellet of human Wharton’s jelly mesenchymal stem cells and rat costal chondrocytes as a candidate for articular cartilage regeneration: in vitro and in vivo study

BackgroundSeeding cells are key factors in cell-based cartilage tissue regeneration. Monoculture of either chondrocyte or mesenchymal stem cells has several limitations. In recent years, co-culture strategies have provided potential solutions. In this study, directly co-cultured rat costal chondrocytes (CCs) and human Wharton’s jelly mesenchymal stem (hWJMSCs) cells were evaluated as a candidate to regenerate articular cartilage.MethodsRat CCs are directly co-cultured with hWJMSCs in a pellet model at different ratios (3:1, 1:1, 1:3) for 21 days. The monoculture pellets were used as controls. RT-qPCR, biochemical assays, histological staining and evaluations were performed to analyze the chondrogenic differentiation of each group. The 1:1 ratio co-culture pellet group together with monoculture controls were implanted into the osteochondral defects made on the femoral grooves of the rats for 4, 8, 12 weeks. Then, macroscopic and histological evaluations were performed.ResultsCompared to rat CCs pellet group, 3:1 and 1:1 ratio group demonstrated similar extracellular matrix production but less hypertrophy intendency. Immunochemistry staining found the consistent results. RT-PCR analysis indicated that chondrogenesis was promoted in co-cultured rat CCs, while expressions of hypertrophic genes were inhibited. However, hWJMSCs showed only slightly improved in chondrogenesis but not significantly different in hypertrophic expressions. In vivo experiments showed that all the pellets filled the defects but co-culture pellets demonstrated reduced hypertrophy, better surrounding cartilage integration and appropriate subchondral bone remodeling.ConclusionCo-culture of rat CCs and hWJMSCs demonstrated stable chondrogenic phenotype and decreased hypertrophic intendency in both vitro and vivo. These results suggest this co-culture combination as a promising candidate in articular cartilage regeneration.

Chondrogenic Characteristics of Auricular Chondrocytes Cocultured With Adipose-Derived Stem Cells are Superior to Stromal Vascular Fraction of Adipose Tissue.

Reconstruction of craniofacial cartilage defects is among the most challenging operations in facial plastic surgery. The co-culture system of partial replacement of chondrocytes by stem cells has been confirmed effective in the repair of cartilaginous defects. The aim of this study is to compare chondrogenic properties of expanded adipose-derived stem cells (ADSCs) and stromal vascular fraction (SVF), including ADSCs/SVF monoculture and coculture with rabbit auricular chondrocytes (ACs). Analysis of morphology, histology, real-time polymerase chain reaction and glycosaminoglycans (GAG) quantification were performed to characterize the chondrogenesis of pellets. The triple differentiation potential of ADSCs had been confirmed. Further, using flow cytometry, the authors demonstrated that ADSCs and SVF have different characteristics in cell surface markers, and ADSCs are more enriched in cells from the mesenchymal lineage than SVF. GAG production of ADSCs is significantly higher than that of SVF in pellet monoculture, and pellet coculture of ADSCs and ACs are better in depositing cartilage matrix than the mixture of SVF and ACs. Our study suggests that ADSCs may be more suitable seed cells for craniofacial cartilage defect or deformity repair.

Co-Culture of Adipose-Derived Stem Cells and Chondrocytes With Transforming Growth Factor-Beta 3 Promotes Chondrogenic Differentiation.

Tissue engineering cartilage is a promising strategy to reconstruct the craniofacial cartilaginous defects. It demands plenty of chondrocytes to generate human-sized craniofacial frameworks. Partly replacement of chondrocytes by adipose-derived stem cells (ADSCs) can be an alternative strategy.The study aimed at evaluating the chondrogenic outcome of ADSCs and chondrocytes in direct co-culture with transforming growth factor-beta (TGF-β3). Porcine ADSCs and chondrocytes were obtained from abdominal wall and external ears. Four groups: ADSCs or chondrocytes monocultured in medium added with TGF-β3; ADSCs and ACs co-cultured with or without TGF-β3. Cell growth rate was performed to evaluate the cell proliferation. Morphological, histologic and real-time polymerase chain reaction analysis were performed to characterize the chondrogenic outcome of pellets. ADSCs had favorable multi-lineage differentiation potential. Further, when ADSCs were co-cultured with chondrocytes in medium added with TGF-β3, the cell proliferation was promoted and the chondrogenic differentiation of ADSCs was enhanced. We demonstrate that pellet co-culture of ADSCs and chondrocyte with TGF-β3 could construct high quantity cartilages. It suggests that this strategy might be useful in future cartilage repair.

Optimizing 3D Co-culture Models to Enhance Synergy Between Adipose-Derived Stem Cells and Chondrocytes for Cartilage Tissue Regeneration

Donor cell scarcity is a key barrier for clinical translation of chondrocytes for regenerating cartilage. To overcome this challenge, recent studies have utilized mixed populations of chondrocytes and adult stem cells to show synergistic interactions during 3D co-culture using hydrogels or pellets. Yet, how different co-culture models impact the synergy and resulting cartilage formation remains unknown. To determine the optimal delivery method of a mixed population of stem cells and chondrocytes for cartilage regeneration, here we compared hydrogel and pellet co-culture on the interactions between adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). While both co-culture models supported synergy, hydrogel co-culture led to over a 5-fold increase in synergistic index in cell proliferation, and over 11 and 15-fold increases in synergistic indices for production of cartilage matrix (collagen and sGAG respectively). Interaction index analyses of gene expressions showed hydrogel co-culture significantly reduced hypertrophic phenotype of both ADSCs and NChons. In hydrogel co-culture, NChons contributed to the neocartilage deposition while ADSCs resided outside the newly formed cartilage nodule, serving as supporting cells through paracrine signaling. In contrast, during pellet co-culture, both NChons and ADSCs contributed to the newly formed cartilage. Using the same number of cells, hydrogel co-culture supported higher synergy and produced neocartilage tissues approximately 5 times the size of pellet co-culture. In contrast, pellet co-culture resulted in denser cartilage matrix suitable only for small defects. The outcomes of this study suggest hydrogel delivery as a more advantageous co-culture method for regenerating cartilage using mixed cell populations. Donor cell scarcity is a key barrier for clinical translation of cell-based therapies for cartilage regeneration. To overcome this challenge, here we harnessed abundantly available fat-derived stem cells and mixed them with juvenile chondrocytes, a cell type from native cartilage that can produce robust cartilage. We compared two different co-culture methods, hydrogel versus cell pellet, to grow the mixed cell populations for cartilage regeneration. We demonstrate that 3D hydrogels induced higher synergy using mixed cell populations, allowing cartilage repair with a reduced number of chondrocytes and defect filling of larger volumes than pellets. Future work will validate the potential of using hydrogels to deliver mixed population of fat-derived stem cells and juvenile chondrocytes for cartilage regeneration in vivo using relevant animal models.

Co-implantation of bone marrow mesenchymal stem cells and chondrocytes increase the viability of chondrocytes in rat osteo-chondral defects.

Replacement of chondrocytes by adult stem cells was believed to improve the performance of autologous chondrocytes transplantation, since less chondrocytes were needed. Previous studies have demonstrated that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) is due to the trophic effects of the MSC by stimulating chondrocyte proliferation and matrix production. However, the destination of MSCs or chondrocytes after implanted in osteo-chondral defects is not clear. The aim of the present study is to investigate the viability of MSCs and chondrocytes after co-implantation into a rat osteo-chondral defect model. MSCs were isolated from bone marrow and chondrocytes were extracted from knee joints of neonatal rats. Results of sulfated glycosaminoglycans (GAG) and collagen quantification demonstrated that co-culture pellets of BMSCs and chondrocytes have more GAG deposition than that of BMSCs or chondrocytes alone. Tracking cells with fluorescence protein demonstrated that MSCs disappeared following co-culture. In a rat knee injury model, co-implantation of BMSCs and chondrocytes contained more viable chondrocytes than chondrocytes implanted alone. To conclude, BMSCs were replaced by chondrocytes in pellet co-culture and BMSCs increased the viability of chondrocytes following co-implantation in a osteo-chondral defects model. Co-implantation of BMSCs and chondrocytes may be a promising approach to repairing osteo-chondral defects in the clinical setting.

Pellet coculture of osteoarthritic chondrocytes and infrapatellar fat pad-derived mesenchymal stem cells with chitosan/hyaluronic acid nanoparticles promotes chondrogenic differentiation

BackgroundCell source plays a key role in cell-based cartilage repair and regeneration. Recent efforts in cell coculture have attempted to combine the advantages and negate the drawbacks of the constituent cell types. The aim of this study was to evaluate the chondrogenic outcome of articular chondrocytes (ACs) and infrapatellar fat pad (IPFP)-derived mesenchymal stem cells (MSCs) in direct coculture.MethodsACs and IPFP MSCs from the same patients with knee osteoarthritis (OA) were cocultured in monolayer and in pellets. The monocultures of each cell type were also used as controls. Morphological and histologic analysis, immunofluorescence staining, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay were performed to characterize the chondrogenic differentiation of cocultures. Furthermore, the effects of chitosan/hyaluronic acid (CS/HA) nanoparticle exposure on the chondrogenesis of cocultures were examined.ResultsIn both monolayer and pellet coculture, the hypertrophy of MSCs and the inflammatory activities of ACs were inhibited, although the chondrogenic production in coculture was not promoted compared with that in monoculture. In addition, the exposure of CS/HA nanoparticles to pellet coculture improved the production of type II collagen and aggrecan.ConclusionsWe demonstrate for the first time that pellet coculture of ACs and IPFP MSCs with CS/HA nanoparticles could promote chondrogenic outcome while preventing the inflammatory status of ACs and the hypertrophic differentiation of MSCs. These findings suggest that the combination of ACs, IPFP MSCs, and CS/HA might be useful in cartilage repair in knee OA.

Chondrocytes Cocultured with Stromal Vascular Fraction of Adipose Tissue Present More Intense Chondrogenic Characteristics Than with Adipose Stem Cells.

Partial replacement of chondrocytes by stem cells has been proposed to improve the performance of autologous chondrocyte implantation (ACI). Our previous studies showed that the increased cartilage production in pellet cocultures of chondrocytes and mesenchymal stem cells (MSCs) is due to a trophic role of the MSCs by stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. The aim of this study is to compare the trophic effects of stromal vascular fraction cells (SVF) and in vitro expanded adipose stem cells (ASC). SVF and culture-expanded ASC (n = 9) were cocultured with primary human chondrocytes in pellets. By glycosaminoglycan (GAG) and DNA assays, we showed that coculture pellets of SVF and chondrocytes have more GAG deposition than that of ASC and chondrocytes. Results of the short tandem repeats analysis indicated that the increase in the chondrocyte proportion in the coculture pellets is more pronounced in the SVF coculture group than in the ASC coculture group. Using flow cytometry and microarray, we demonstrated that SVF and ASC have different characteristics in cell surface markers and gene expression profiles. SVF is more heterogeneous than ASC, whereas ASC is more enriched in cells from the mesenchymal lineage than SVF. By subcutaneous implantation into nude mice, we showed that constructs of SVF and chondrocytes are better in depositing cartilage matrix than the mixture of ASC and chondrocytes. Taken together, SVF is better than ASC in terms of forming cartilage matrix in pellet coculture and in coimplantation models omitting the need for prior cell expansion. Our study suggests that the SVF in combination with primary human chondrocytes may be a good cell combination for one-stage cartilage repair.

Adventitial Cells and Perictyes Support Chondrogenesis Through Different Mechanisms in 3-Dimensional Cultures With or Without Nanoscaffolds.

In previous studies, mesenchymal stromal cells (MSCs) derived from bone marrow and fat tissues were shown to increase proliferation and matrix production of chondrocytes (CH) in co-culture. The aim of this study was to investigate the roles of pericytes (CD31(neg)CD45(neg)CD146+CD34(neg)) and adventitial cells (CD31(neg)CD45(neg)CD146(neg)CD34+) sub-populations of MSCs in supporting proliferation and matrix deposition of CH. The MSCs were derived from synovial membrane and attaching fat tissue. Then, the pericytes and adventitial cells were sorted from total MSCs and co-cultured with articular CH respectively. In pellet co-culture model, the pericytes showed more prominent effects on glycosaminoglycans (GAGs) production and Collagen II synthesis than the adventitial cells which had stronger effects on promoting CH proliferation. In addition, quantitative polymerase chain reaction (qPCR) was performed to examine the expression of a group of secreted growth factors and co-culture performed on electrospun scaffolds based on Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), to verify the trophic effects of different MSC sub-populations in 3-Dimensional (3D) environment. In conclusion, it was found that the pericytes and adventitial cells support CH in different ways; the adventitial cells more supporting the proliferation of CH, while pericytes are better in stimulating GAGs and collagen production of CH.

Beneficial effects of coculturing synovial derived mesenchymal stem cells with meniscus fibrochondrocytes are mediated by fibroblast growth factor 1: increased proliferation and collagen synthesis.

Meniscus reconstruction is in great need for orthopedic surgeons. Meniscal fibrochondrocytes transplantation was proposed to regenerate functional meniscus, with limited donor supply. We hypothesized that coculture of synovial mesenchymal stem cells (SSC) with meniscal fibrochondrocytes (me-CH) can support matrix production of me-CH, thus reducing the number of me-CH needed for meniscus reconstruction. A pellet coculture system of human SSC and me-CH was used in this study. Enhanced glycosaminoglycans (GAG) in coculture pellets were demonstrated by Alcian blue staining and GAG quantification, when compared to monoculture. More collagen synthesis was shown in coculture pellets by hydroxyproline assay. Increased proliferation of me-CH was observed in coculture. Data from BrdU staining and ELISA demonstrated that conditioned medium of SSCs enhanced the proliferation and collagen synthesis of me-CH, and this effect was blocked by neutralizing antibody against fibroblast growth factor 1 (FGF1). Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK). Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1. Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus.

Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells.

Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe the methods for making coculture pellets of MSCs and chondrocytes. We also provide detailed protocols for analyzing coculture pellets with cell tracking, proliferation assays, species specific polymerase chain reactions (PCR), short tandem repeats analysis, and histological examination.

Periodontal ligament mesenchymal stromal cells increase proliferation and glycosaminoglycans formation of temporomandibular joint derived fibrochondrocytes.

Objectives. Temporomandibular joint (TMJ) disorders are common disease in maxillofacial surgery. The aim of this study is to regenerate fibrocartilage with a mixture of TMJ fibrochondrocytes and periodontal ligament derived mesenchymal stem cells (PD-MSCs). Materials and Methods. Fibrochondrocytes and PD-MSC were cocultured (ratio 1 : 1) for 3 weeks. Histology and glycosaminoglycans (GAGs) assay were performed to examine the deposition of GAG. Green florescent protein (GFP) was used to track PD-MSC. Conditioned medium of PD-MSCs was collected to study the soluble factors. Gene expression of fibrochondrocytes cultured in conditioned medium was tested by quantitative PCR (qPCR). Results. Increased proliferation of TMJ-CH was observed in coculture pellets when compared to monoculture. Enhanced GAG production in cocultures was shown by histology and GAG quantification. Tracing of GFP revealed the fact that PD-MSC disappears after coculture with TMJ-CH for 3 weeks. In addition, conditioned medium of PD-MSC was also shown to increase the proliferation and GAG deposition of TMJ-CH. Meanwhile, results of qPCR demonstrated that conditioned medium enhanced the expression levels of matrix-related genes in TMJ-CH. Conclusions. Results from this study support the mechanism of MSC-chondrocyte interaction, in which MSCs act as secretor of soluble factors that stimulate proliferation and extracellular matrix deposition of chondrocytes.

Trophic stimulation of articular chondrocytes by late‐passage mesenchymal stem cells in coculture

Coculture of mesenchymal stem cells (MSCs) with articular chondrocytes (ACs) increases glycosaminoglycan (GAG) accumulation compared to monoculture. MSCs might (1) differentiate into ACs (progenitor role) and/or (2) stimulate AC matrix metabolism (trophic role). MSCs lose the ability to undergo chondrogenesis after extended passaging. We hypothesized that MSCs act predominantly as progenitors, and that late-passage MSCs without chondrogenic potential would be unable to increase GAG in coculture. Early- and late-passage human MSCs (hMSCs) were grown in pellet monoculture under chondrogenic conditions and in pellet coculture with bovine ACs. Chondrogenesis was assessed with GAG quantification, safranin-O staining, and quantitative PCR (qPCR). Contributions of human and bovine cells were assessed with species-specific qPCR and human-specific immunostaining. Late-passage hMSCs did not undergo chondrogenesis in monoculture with chondrogenic stimuli or in coculture with ACs. Early-passage hMSCs underwent chondrogenesis only in response to chondrogenic stimuli. Coculture pellets in both cases accumulated as much GAG/DNA as monoculture AC pellets. Aggrecan transcription was not increased in coculture. Late-passage hMSCs that do not undergo chondrogenesis are capable of stimulating GAG accumulation in coculture with ACs. This supports a trophic effect of hMSCs on ACs. hMSCs may have therapeutic utility even after prolonged passaging.

Fibroblast growth factor-1 is a mesenchymal stromal cell-secreted factor stimulating proliferation of osteoarthritic chondrocytes in co-culture.

Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis patients were co-cultured with human bone marrow-derived MSCs (hMSCs) in pellets. Genome-wide mRNA expression analysis and quantitative polymerase chain reactions (qPCR) were used to identify soluble factors that were specifically induced in co-cultures. Immunofluorescent staining combined with cell tracking and enzyme-linked immunosorbent assay (ELISA) were performed to validate up-regulation at the protein level and to identify the cellular origin of the increased proteins. Chemical blockers and neutralizing antibodies were used to elucidate the role of the identified candidate genes in co-cultures. A number of candidate factors were differentially regulated in co-cultures at the mRNA level. Of these, fibroblast growth factor-1 (FGF-1) mRNA and protein expression were markedly increased in co-cultures predominantly due to up-regulated expression in MSCs. Blocking of FGF signaling in co-culture pellets by specific FGF receptor inhibitors or FGF-1 neutralizing antibodies completely blocked hPCs proliferation. We demonstrate that MSCs increase FGF-1 secretion on co-culture with hPCs, which, in turn, is responsible for increased hPCs proliferation in pellet co-cultures.

Abstract 231: Bone Marrow-Derived Mesenchymal Stem Cells from Amputated Limbs of Patients with Critical Limb Ischemia Provide Stromal Support for Endothelial Cells

Critical limb ischemia (CLI) often results in amputation, but autologous cellular therapy using bone marrow-derived mesenchymal stem cells (MSCs) may be able to prevent this outcome. However, the quality of MSCs in the CLI population is not clear. Amputated CLI limbs contain reservoirs of MSCs that can be used to characterize their stromal capacity. We hypothesize that MSC stromal capacity is relatively preserved in ischemic limbs compared to ones from healthy donors. Methods Healthy donor MSCs (hdMSCs, n=2) and ischemic MSCs (iMSCs, n=3) from amputated limbs of non-diabetic CLI patients were cultured. Human microdermal ECs were commercially obtained. Co-culture pellets (1:1 ratio) of hdMSC:EC, iMSC:EC, and EC alone were formed and cultured in 3-D fibrin hydrogels in EC growth media. Images of the pellets and invasion areas were captured up to 5 days. The invasion area was quantified as the difference between the total and pellet area normalized to initial pellet area. Experiments were performed in quadruplicates and co-culture experiments were duplicated. Results iMSC:EC co-culture provided similar stromal support as the healthy donor cohort. Both MSC groups were significantly greater than the ECs alone following day 2 (see figure, *P<0.0005, ANOVA). Both MSC groups also exhibited significant increase in invasion area with time (P<0.01) while the ECs did not (P>0.5). Conclusion This is the first study to demonstrate that iMSCs provide similar stromal support to ECs as healthy hdMSCs, thus iMSCs may be of sufficient quality for use in autologous therapy. We are currently quantifying secretome expressions of the iMSCs to provide insights into mechanisms of the stromal support.

Fibroblast growth factor-1 is a mesenchymal stromal cell secreted factor stimulating proliferation of osteoarthritic chondrocytes

Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis patients were co-cultured with human bone marrow-derived MSCs (hMSCs) in pellets. Genome-wide mRNA expression analysis and quantitative polymerase chain reactions (qPCR) were used to identify soluble factors that were specifically induced in co-cultures. Immunofluorescent staining combined with cell tracking and enzyme-linked immunosorbent assay (ELISA) were performed to validate up-regulation at the protein level and to identify the cellular origin of the increased proteins. Chemical blockers and neutralizing antibodies were used to elucidate the role of the identified candidate genes in co-cultures. A number of candidate factors were differentially regulated in co-cultures at the mRNA level. Of these, fibroblast growth factor-1 (FGF-1) mRNA and protein expression were markedly increased in co-cultures predominantly due to up-regulated expression in MSCs. Blocking of FGF signaling in co-culture pellets by specific FGF receptor inhibitors or FGF-1 neutralizing antibodies completely blocked hPCs proliferation. We demonstrate that MSCs increase FGF-1 secretion on co-culture with hPCs, which, in turn, is responsible for increased hPCs proliferation in pellet co-cultures.

The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair

Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

Structured Bilaminar Coculture Outperforms Stem Cells and Disc Cells in a Simulated Degenerate Disc Environment

This study explores the use of bilaminar coculture pellets of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. The pellets were tested under conditions that mimic the degenerative disc. Our goal was to optimize our cell-based therapy in vitro under conditions representative of the eventual diseased tissue. Harnessing the potential of stem cells is an important strategy for regenerative medicine. Our approach directed the behavior of stem cells by mimicking embryonic processes underlying cartilage and intervertebral disc development. Prior experiments have shown that bilaminar coculture can help differentiate MSC and substantially improve new matrix deposition. We have designed a novel spherical bilaminar cell pellet (BCP) where MSCs are enclosed in a shell of NPC. There were 3 groups: MSC, NPC, and BCP. The pellets were tested under 3 different culture conditions: 1) in a bioreactor that provides pressure and hypoxia (mimicking normal disc conditions): 2) with inflammatory cytokines (IL-1b and TNF-a); and 3) a bioreactor with inflammation (mimicking painful disc conditions). When cultured in the bioreactor, the NPC pellets produced significantly more glycosaminoglycans (GAGs) per cell than the other groups: 70% to 80% more than the BCP and the MSC alone. When cultured in an inflammatory environment, the MSC and BCP groups produced 30% to 34% more GAGs per cell than NPC (P < 0.05). When the pellets were cultured in a bioreactor with inflammation, the BCP made 25% more GAGs per cell than the MSC and 57% more than the NPC (P < 0.05). This study shows that BCPs outperform controls in a simulated degenerated disc environment. Adapting inductive mechanisms from development to trigger differentiation and restore diseased tissue has many advantages. As opposed to strategies that require growth factor supplements or genetic manipulations, our method is self-sustaining, targeted, and minimally invasive injection.

Trophic Effects of Mesenchymal Stem Cells in Chondrocyte Co-Cultures are Independent of Culture Conditions and Cell Sources

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. These studies were performed in a culture medium that was not compatible with the chondrogenic differentiation of MSCs. In this study, we tested whether the trophic role of the MSCs is dependent on culturing co-culture pellets in a medium that is compatible with the chondrogenic differentiation of MSCs. In addition, we investigated whether the trophic role of the MSCs is dependent on their origins or is a more general characteristic of MSCs. Human BM-MSCs and bovine primary chondrocytes were co-cultured in a medium that was compatible with the chondrogenic differentiation of MSCs. Enhanced matrix production was confirmed by glycosaminoglycans (GAG) quantification. A species-specific quantitative polymerase chain reaction demonstrated that the cartilage matrix was mainly of bovine origin, indicative of a lack of the chondrogenic differentiation of MSCs. In addition, pellet co-cultures were overgrown by bovine cells over time. To test the influence of origin on MSCs' trophic effects, the MSCs isolated from adipose tissue and the synovial membrane were co-cultured with human primary chondrocytes, and their activity was compared with BM-MSCs, which served as control. GAG quantification again confirmed increased cartilage matrix production, irrespective of the source of the MSCs. EdU staining combined with cell tracking revealed an increased proliferation of chondrocytes in each condition. Irrespective of the MSC source, a short tandem repeat analysis of genomic DNA showed a decrease in MSCs in the co-culture over time. Our results clearly demonstrate that in co-culture pellets, the MSCs stimulate cartilage formation due to a trophic effect on the chondrocytes rather than differentiating into chondrocytes, irrespective of culture condition or origin. This implies that the trophic effect of MSCs in co-cultures is a general phenomenon with potential implications for use in cartilage repair strategies.

Structured Coculture of Mesenchymal Stem Cells and Disc Cells Enhances Differentiation and Proliferation

Purpose: During in vivo stem cell differentiation, mature cells often induce the differentiation of nearby stem cells. Accordingly, prior studies indicate that a randomly mixed coculture can help transform mesenchymal stem cells (MSC) into nucleus pulposus cells (NPC). However, because in vivo signaling typically occurs heterotopically between adjacent cell layers, we hypothesized that a structurally organized coculture between MSC and NPC will result in greater cell differentiation and proliferation over single cell-type controls and cocultures with random organization. Methods: We developed a novel bilaminar cell pellet (BCP) system where a sphere of MSC is enclosed in a shell of NPC by successive centrifugation. Controls were made using single cell-type pellets and coculture pellets with random organization. The pellets were evaluated for DNA content, gene expression, and histology. Results: A bilaminar 3D organization enhanced cell proliferation and differentiation. BCP showed significantly more cell proliferation than pellets with one cell type and those with random organization. Enhanced differentiation of MSC within the BCP pellet relative to single cell-type pellets was demonstrated by quantitative RT-PCR, histology, and in situ hybridization. Conclusions: The BCP culture system increases MSC proliferation and differentiation as compared to single cell type or randomly mixed coculture controls.

Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM-MSC) or adipose tissue (AT-MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM-MSC with bovine AC, (ii) BM-MSC from HLA-A2+ with AC from HLA-A2- donors, or (iii) human green fluorescent protein transduced BM-MSC with AC. Human BM-MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT-PCR. Coculture of NC or AC (25%) with BM-MSC or AT-MSC (75%) in pellets resulted in up to 1.6-fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM-MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2-fold) if cocultured with BM-MSC and maintained a differentiated phenotype. Chondro-induction in MSC-chondrocyte coculture is a robust process mediated by two concomitant effects: MSC-induced chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair.