Plastic Surgery Research Council

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Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Co-Culture with Xenogeneic Swine Chondrocytes
Yingfang Fan, M.D., Ph.D., Gem Runyan, BS, Samuel D. Zarfos, BS, Mark A. Randolph, MAS.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE: Cartilage has very limited ability for self repair and regeneration. Although autologous chondrocytes can be obtained from uninvolved areas of the joint to re-implant and repair lesions, the additional joint morbidity and costly in vitro expansion of primary chondrocytes limits widespread clinical application. Human bone marrow mesenchymal stem cells (hBMSCs) can be obtained by minimally invasive methods and could be a favorable source for cartilage regeneration. However, controlling the differentiation of BMSCs into the chondrocyte phenotype has hampered clinical implementation. The stimulation of human hBMSCs with xenogeneic chondrocytes has great potential for increasing the numbers of chondrogenic cells for cellular cartilage repair. The aim of this study was to determine the differentiation effects on human BMSCs and swine BMSc (sBMSc) by co-culture with swine chondrocytes or culture medium conditioned by chondrocytes.
METHODS: Swine articular chondrocytes were isolated and grown in standard chondrocyte culture medium that was collected and used as chondrocyte-conditioned medium (CCM). hBMSCs and sBMSCs were expanded in normal bone marrow medium for 3 weeks and transferred to 6 well plates and Transwell plates for an additional one week. Then BMSCs cultured in 6-well plates were treated with CCM for 10 days. In the transwell cultures swine articular chondrocytes (sACs) were added in co-culture with the BMSCs for 10 days. Four different groups were performed: (1) hCCM, (2) hTranswell, (3) sCCM, (4) sTranswell. Real-time polymerase chain reaction was performed to evaluate the gene expression of collagen type II (COL2A1), and aggrecan (ACAN).
RESULTS: After 10 days of culture, COL2A1 gene expression in hTranswell group was significantly increased 20-fold (p < 0.00001), compared with hCCM group, indicating that co-culture with chondrocytes had a greater effect on chondrogenic differentiation than the conditioned medium alone. ACAN expression in hTranswell group was significantly higher than hCCM group (p = 0.003), which further demonstrated that xenogeneic swine chondrocytes have a strong effect on chondrogenic differentiation of hBMSCs. ACAN gene expression in sTranswell group showed 30-fold greater than sCCM group (p = 0.00022), indicating this autologous co-culture model has potential to differentiate both hBMSC and sBMSC towards a chondrogenic phenotype.
CONCLUSION: These results indicate that both xenogeneic co-culture models are capable differentiating hBMSC towards a chondrogenic phenotype. This approach could eliminate the need for autologous chondrocyte harvest by using a plentiful supply of xenogeneic chondrocytes to chondrodifferentiate a patient's own MSCs for cartilage repair. Recent development of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine may even provide a source of xenogeneic MSCs that can be used in humans without the consequence of immune rejection.


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