間質幹細胞 (mesenchymal stem cells, MSCs) 被期待作為軟骨疾病新世代細胞治療的良好細胞來源，但由於不佳的分化效率以及會產生非軟骨組織分化，使得在臨床應用上仍然無法獲得進一步突破，如何改進MSCs軟骨分化便是突破此瓶頸的關鍵。傳統上以TGFβ作為MSCs體外培養軟骨分化之標準誘導劑，然而TGFβ為多能誘導因子，可促使多種類型細胞分化而並不限於軟骨，且實驗發現於軟骨誘導條件下仍會產生脫靶效應而降低軟骨分化效率及移植副作用。
本研究證實運用Wnt/β-catenin拮抗劑，可以在不施加TGFβ情況下更有效率地誘導多種來源MSCs (分離自成體組織或分化自多能幹細胞) 的軟骨分化，並減少細胞分化往非目標軟骨細胞，如平滑肌細胞或肥大化軟骨細胞等。Wnt/β-catenin拮抗劑除了透過增強N-cadherin表現量與N-cadherin-β-catenin交互作用強化黏著連接 (adherens junctions, AJs) ，增進細胞骨架驅使之凝縮作用(condensation) 加快軟骨分化進程，也透過降低細胞核內β-catenin表現量而降低成骨分化或軟骨細胞肥大化之作用。除了體外細胞培養與動物實驗驗證外，我們也透過轉錄體分析證實Wnt/β-catenin拮抗劑對比傳統TGFβ方式降低脫靶分化；於人類初代細胞 (primacy cells) 比較中更發現軟骨細胞比起MSCs或成骨細胞具有更低之TGFβ與Wnt/β-catenin訊息表現，而具有更高之α-catenin/AJ表現，顯示我們提出以Wnt/β-catenin拮抗劑取代TGFβ進行軟骨分化更貼近真實軟骨組織細胞之生理情況。
此研究揭示了透過對發育學上的基礎訊息表現背景的了解以及調控細胞結構互動作用能夠達成更有效的軟骨分化，並具有更高之轉譯與臨床應用潛力。

Mesenchymal stem cells (MSCs) are expected as superior cell source for new generation of cell therapy treating cartilage diseases. However, the poor chondrogenic efficiency and the non-chondrogenic lineage differentiation limits its progress in application. Improvement of MSC chondrogenic differentiation is urgently needed to break through the bottleneck. However, MSC chondrogenesis is commonly induced through TGFβ, a pleomorphic growth factor without specificity for this lineage. In this study, utilizing tissue- and induced pluripotent stem cell-derived MSCs, we demonstrate an efficient and precise approach to induce chondrogenesis through Wnt/β-catenin antagonism alone without TGFβ. Compared to TGFβ, Wnt/β-catenin antagonism more rapidly induced MSC chondrogenesis without eliciting off-target lineage specification towards smooth muscle or hypertrophy; this was mediated through increasing N-cadherin levels and β-catenin interactions—key components of the adherens junctions (AJ)—and increasing cytoskeleton-mediated condensation. Downregulation of off-target lineage commitment was confirmed by mRNA expression profiles of chondrogenic MSCs treated by Wnt antagonist. Validation with transcriptomic analysis of human chondrocytes compared to MSCs and osteoblasts showed significant downregulation of Wnt/β-catenin and TGFβ signaling along with upregulation of α-catenin- and AJ-related processes. Our findings underscore the importance of understanding developmental pathways and structural modifications in achieving efficient MSC chondrogenesis for translational application.

摘要----------------------------ii
ABSTRACT---------------------iii
致謝 ---------------------------iv
INTRODUCTION----------------1
MATERIALS AND METHODS----8
RESULTS----------------------14
DISCUSSION------------------25
FIGURES AND TABLES-------- 34
REFERENCES------------------71

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