以幾丁聚醣/玻尿酸生物材料將誘導式多能幹細胞分化為軟骨細胞

Abstract

骨關節炎（osteoarthritis）是老年常見的疾病，造成病患膝關節、臀關節、指關節等部位發炎腫痛，影響行動與生活品質。目前治療骨關節炎以給予抑制發炎藥物與物理治療為主，然而隨著再生醫學技術的進展，細胞治療可望成為骨關節炎的治療選項之一。以細胞治療骨關節炎，可注入病患自身的軟骨細胞，或者移植原位間質幹細胞（primary mesenchymal stem cells）分化而來的軟骨細胞，但這些方法分別有軟骨細胞難以體外培養放大、侵入性取得間質幹細胞易傷害宿主等問題。利用誘導式多功能幹細胞（induced pluripotent stem cells），之後體外分化為軟骨細胞再移植骨關節炎病患，是可行的替代方案之一。然而，將誘導式多能幹細胞分化為間質幹細胞，再分化為軟骨細胞的過程漫長，且間質幹細胞分化而來的軟骨易成熟肥大，限制其修復關節軟骨的能力。另一方面，若將誘導式多能幹細胞形成的類胚體（embryoid bodies）直接分化為軟骨細胞，此軟骨細胞不易肥大，且分化時程較間質幹細胞來源的軟骨短，因此更具細胞治療價值。 本研究參考過往文獻，測試自行配置的軟骨生成定義液，和商業販售的軟骨分化培養液效果一致，能將人類誘導式多能幹細胞經過類胚體，藉由「向外生長法（EB-outgrowth）」直接分化成軟骨細胞。此軟骨細胞能分泌胞外聚集蛋白聚醣（aggrecan）、第二型膠原蛋白（type II collagen），並表現軟骨相關基因，如SOX9、COL2A1、ACAN。此外，為了應用軟骨細胞治療於臨床前的模式生物兔子（Oryctolagus cuniculus），本研究也以此套分化法，將兔子的誘導式多能幹細胞分化成軟骨細胞。此結果顯示，該分化法能於不同物種上作用，即便人類和兔子誘導式多能幹細胞有不同的細胞型態。最後，由於體內軟骨細胞生長於富含聚集蛋白聚醣和第二型膠原蛋白的微環境中，推測在體外分化過程中，給予軟骨聚集蛋白聚醣組成之一的玻尿酸（hyaluronic acid），可幫助誘導式多能幹細胞—類胚體（iPSC-EBs）更有效的分化為軟骨細胞。因此，本篇將誘導式多能幹細胞—類胚體於幾丁聚醣/玻尿酸的膜上分化，以同樣的分化方式，得到表現較多軟骨生成基因如第二型膠原蛋白，且表現較少成熟肥大基因如COL10A1的軟骨細胞，證明分化環境對於軟骨細胞之重要性。綜上所述，本研究證明除了人類誘導式多能幹細胞，兔子誘導式多能幹細胞也能分化為軟骨細胞，且於玻尿酸上分化軟骨，有助於提升體外軟骨的分化效率。

Osteoarthritis (OA) is a prevalent disease in aged people, causing pain of knee joints, hip joints and finger joints so that affect action and quality of life. Currently symptomatic treatments of anti-inflammatory drugs and physical therapy are the main therapy of OA. However, as stem cell technology develops, cell therapy is expected to become an option for OA therapy. Patient-derived chondrocytes or mesenchymal stem cells (MSCs) are autologous cell resources of OA cell therapy. However, primary chondrocytes are difficult to reserve in vitro and MSCs are hard to maintain on high stem cell activity and proliferative index in vitro. Induced pluripotent stem cells (iPSCs) are ideal cell sources to generate chondrocytes in vitro. Induced pluripotent stem cells (iPSCs) are ideal cell sources to generate chondrocytes in vitro. iPSCs could be differentiated into chondrocytes directly or via iPSC-derived MSCs. Compared to differentiation of iPSC-MSC-chondrocytes, the directly differentiation method needs less differentiation time and results in lower levels of cell hypertrophy and maturation. First, we chose a system which directly differentiated human iPSC into chondrocytes in the chondrogenesis-defined medium by the EB-outgrowth method based on previous studies. The differentiation potency of the chondrogenesis-defined medium was the same as the commercial chondrogenic medium. The iPSC-derived chondrocytes had ability to secret proteoglycans and type II collagens, and also expressed chondrogenic genes such as SOX9, COL2A1 and ACAN. Besides human iPSCs, for use of the preclinical model animal Oryctolagus cuniculus (rabbit), we also differentiated rabbit iPSCs into chondrocytes by this system. It suggests the system could be used on iPSCs of different species, even though rabbit iPSCs have different phenotypes to human iPSCs. Furthermore, we tried to differentiate iPSCs into chondrocytes on the membrane grafted by hyaluronic acid (HA), one of components of a chondrogenic ECM. This result showed that iPSC-EBs differentiated to chondrocytes on HA exhibited higher levels of chondrogenic markers such as COL2A1 and lower levels of hypertrophic markers such as COL10A1. In conclusion, this study demonstrates not only human iPSCs but also rabbit iPSCs could be differentiated into chondrocytes, and chondrogenic induction on HA enhances efficiency of chondrogenesis in vitro.