CD49f and CD146: A Possible Crosstalk Modulates Adipogenic Differentiation Potential of Mesenchymal Stem Cells.

BACKGROUND: The lack of appropriate mesenchymal stem cells (MSCs) selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs. Genetic engineering coupled with high-throughput proteomic studies of MSC surface markers arises as a promising strategy to identify stem cell-specific markers. However, the technical limitations are the key factors making it less suitable to provide an appropriate starting material for the screening platform. A more accurate, easily accessible approach is required to solve the issues. METHODS: This study established a high-throughput screening strategy with forward versus side scatter gating to identify the adipogenesis-associated markers of bone marrow-derived MSCs (BMSCs) and tonsil-derived MSCs (TMSCs). We classified the MSC-derived adipogenic differentiated cells into two clusters: lipid-rich cells as side scatter (SSC)-high population and lipid-poor cells as SSC-low population. By screening the expression of 242 cell surface proteins, we identified the surface markers which exclusively found in lipid-rich subpopulation as the specific markers for BMSCs and TMSCs. RESULTS: High-throughput screening of the expression of 242 cell surface proteins indicated that CD49f and CD146 were specific for BMSCs and TMSCs. Subsequent immunostaining confirmed the consistent specific expression of CD49f and CD146 and in BMSCs and TMSCs. Enrichment of MSCs by CD49f and CD146 surface markers demonstrated that the simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis of mesenchymal stem cells. Furthermore, the fate decision of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f+CD146+ immunophenotype. CONCLUSIONS: We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins. This provided a valuable tool to investigate MSC-specific markers. Additionally, we showed a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.

Human Fetal Cartilage-Derived Progenitor Cells Exhibit Anti-Inflammatory Effect on IL-1ß-Mediated Osteoarthritis Phenotypes In Vitro.

BACKGROUND: In this study, we have investigated whether human fetal cartilage progenitor cells (hFCPCs) have anti-inflammatory activity and can alleviate osteoarthritis (OA) phenotypes in vitro. METHODS: hFCPCs were stimulated with various cytokines and their combinations and expression of paracrine factors was examined to find an optimal priming factor. Human chondrocytes or SW982 synoviocytes were treated with interleukin-1ß (IL-1ß) to produce OA phenotype, and co-cultured with polyinosinic-polycytidylic acid (poly(I-C))-primed hFCPCs to address their anti-inflammatory effect by measuring the expression of OA-related genes. The effect of poly(I-C) on the surface marker expression and differentiation of hFCPCs into 3 mesodermal lineages was also examined. RESULTS: Among the priming factors tested, poly(I-C) (1 µg/mL) most significantly induced the expression of paracrine factors such as indoleamine 2,3-dioxygenase, histocompatibility antigen, class I, G, tumor necrosis factor- stimulated gene-6, leukemia inhibitory factor, transforming growth factor-ß1 and hepatocyte growth factor from hFCPCs. In the OA model in vitro, co-treatment of poly(I-C)-primed hFCPCs significantly alleviated IL-1ß-induced expression of inflammatory factors such as IL-6, monocyte chemoattractant protein-1 and IL-1ß, and matrix metalloproteinases in SW982, while it increased the expression of cartilage extracellular matrix such as aggrecan and collagen type II in human chondrocytes. We also found that treatment of poly(I-C) did not cause significant changes in the surface marker profile of hFCPCs, while showed some changes in the 3 lineages differentiation. CONCLUSION: These results suggest that poly(I-C)-primed hFCPCs have an ability to modulate inflammatory response and OA phenotypes in vitro and encourage further studies to apply them in animal models of OA in the future.

Development of Surgically Transplantable Parathyroid Hormone-Releasing Microbeads.

Hypoparathyroidism is an endocrine disorder that occurs because of the inability to produce parathyroid hormone (PTH) effectively. Previously, we reported the efficacy of tonsil-derived mesenchymal stem cells (TMSCs) differentiated into parathyroid-like cells for the treatment of hypoparathyroidism. Here, we investigated the feasibility of three-dimensional structural microbeads fabricated with TMSCs and alginate, a natural biodegradable polymer, to treat hypoparathyroidism. Alginate microbeads were fabricated by dropping a 2% (w/v) alginate solution containing TMSCs into a 5% CaCl2 solution and then differentiated into parathyroid-like cells using activin A and sonic hedgehog for 7 days. The protein expression of PTH, a specific marker of the parathyroid gland, was significantly higher in differentiated alginate microbeads with TMSCs (Al-dT) compared with in undifferentiated alginate microbeads with TMSCs. For in vivo experiments, we created the hypoparathyroidism animal model by parathyroidectomy (PTX) and implanted alginate microbeads in the dorsal interscapular region. The PTX rats with Al-dT (PTX+Al-dT) showed the highest survival rate and weight change and a gradual increase in serum intact PTH levels. We also detected a higher expression of PTH in retrieved tissues of PTX+Al-dT using immunofluorescence analysis. This study demonstrates that alginate microbeads are potential a new tool as a surgically scalable therapy for treating hypoparathyroidism.

Development of Acellular Respiratory Mucosal Matrix Using Porcine Tracheal Mucosa.

BACKGROUND: Respiratory mucosa defects result in airway obstruction and infection, requiring subsequent functional recovery of the respiratory epithelium. Because site-specific extracellular matrix (ECM) facilitates restoration of organ function by promoting cellular migration and engraftment, previous studies considered decellularized trachea an ideal ECM; however, incomplete cell removal from cartilage and mucosal-architecture destruction are frequently reported. Here, we developed a decellularization protocol and applied it to the respiratory mucosa of separated porcine tracheas. METHODS: The trachea was divided into groups according to decellularization protocol: native mucosa, freezing-thawing (FT), FT followed by the use of Perasafe-based chemical agents before mucosal separation (wFTP), after mucosal separation (mFTP), and followed by DNase decellularization (mFTD). Decellularization efficacy was evaluated by DNA quantification and hematoxylin and eosin staining, and ECM content of the scaffold was evaluated by histologic analysis and glycosaminoglycan and collagen assays. Biocompatibility was assessed by cell-viability assay and in vivo transplantation. RESULTS: The mFTP mucosa showed low antigenicity and maintained the ECM to form a proper microstructure. Additionally, tonsil-derived stem cells remained viable when cultured with or seeded onto mFTP mucosa, and the in vivo host response showed a constructive pattern following implantation of the mFTP scaffolds. CONCLUSION: These results demonstrated that xenogenic acellular respiratory mucosa matrix displayed suitable biocompatibility as a scaffold material for respiratory mucosa engineering.