以生醫材料調控人類脂肪幹細胞形成球狀體之研究

Abstract

間葉幹細胞 (mesenchymal stem cells)具有迅速增殖和分化成多種類型細胞的特性，因此被認為應用在再生醫學方面有很大的潛力。脂肪幹細胞（adipose-derived stem cells）已被證明與從骨髓中分離的間葉幹細胞具類似特性，而且可藉由抽脂手術大量取得。但是脂肪幹細胞的幹細胞特性，特別是多能分化指標 (pluripotency markers)的表現，卻會於體外培養過程中迅速下降。這個問題將會限制脂肪幹細胞在細胞治療或組織工程方面的應用。文獻報告曾指出以三維細胞球體型態培養之間葉幹細胞可提高其治療潛力。因此，我們的實驗目的是研究培養人類脂肪幹細胞在甲殼素 (chitosan)表面，而引起細胞結為團塊之現象。隨著我們增加播種細胞之密度，脂肪幹細胞球體的形成速度更快，直徑表也更大。活/死細胞染色試驗顯示三維細胞球體內部大部分細胞仍是存活的。而且三維細胞球體培養時，多能分化指標的基因表現，如Sox-2, Oct-4及 Nanog，相較於平面培養皆顯著提高。我們並使用西方點墨法和免疫螢光染色確認人類脂肪幹細胞以三維細胞球體培養時，其多能分化指標有表現增強現象。我們亦發現人類脂肪幹細胞形成三維細胞球體後會具有出較強的再生能力，包括細胞增殖活性和細胞群落形成能力。最後，我們測試人類脂肪幹細胞在三維細胞球體中的分化潛能，在適當的培養環境下可保持誘導脂肪分化、骨分化 (中胚層)的能力，而朝神經 (外胚層)和肝 (內胚層)分化的能力甚至更強。 有鑑於這些令人鼓舞的結果，我們進而設計了複合的甲殼素/明膠薄膜作為提升脂肪幹細胞的轉移及成球效率之生物材料。我們發現75%甲殼素/25%明膠混摻組合之薄膜可用於動態調控細胞，具最有效率轉移脂肪幹細胞的用途，具做為敷料基材或組織修復貼片的潛力，例如可應用於治療皮膚傷口。表皮困難傷口之癒合不良是一棘手的臨床問題，目前的治療方式皆有其限制，幹細胞療法為一具有潛力的新治療方式。脂肪幹細胞可以通過分化及分泌生長因子的機制促進皮膚傷口癒合，而且我們進一步發現人類脂肪幹細胞若在甲殼素膜的表面形成球體後再打散於二維系統中以單層培養，仍會保持其較強的多能分化指標表現 (Sox-2, Oct-4及Nanog)與血管新生相關生長因子生成 (hepatocyte growth factor及vascular endothelial growth factor)。這些經短暫成球處理的脂肪幹細胞並具較高之增殖活性、較低之細胞凋亡率、較高之基質金屬蛋白酶（matrix metalloproteinases)生成，這些現象皆與此類細胞具較高之CXCR4表現有關，而受傷組織釋放之stromal-derived factor-1 (SDF-1)和CXCR4之間的相互作用對間葉幹細胞往損傷部位歸巢 (homing)扮演著重要的角色。此外，在傷口模式的動物實驗中，施以脂肪幹細胞球體打散再培養的細胞治療會比以持續單層培養的脂肪幹細胞更加強傷口癒合，而其加強傷口癒合的機制來自較高的脂肪幹細胞存活率、較強的幹細胞分化潛能與血管新生能力。綜上所述，以生醫材料調控人類脂肪幹細胞形成球狀體確有其施用於臨床上幫助組織修復之價值。

Because of their abilities of rapid proliferation and differentiation into multiple cell types, mesenchymal stem cells are regarded to have great potentials for application in regenerative medicine. The adipose-derived stem cells (ASCs) have been shown to share similar characteristics of mesenchymal stem cells isolated from bone marrow, and they can be harvested in large amount by liposuction. However, the stem cell characteristics of ASC, indicated by the expression of pluripotency markers, quickly decreased during in vitro culture. This problem has limited the application of ASCs in cell therapy or tissue engineering. Previous reports have suggested that culture as three-dimensional spheroids can increase therapeutic potentials of mesenchymal stem cells. Therefore, we aimed to manipulate the spheroid formation of human ASCs by culturing them on chitosan films. With the increasing seeding density of ASCs on chitosan films, we found that ASC spheroids formed faster and exhibited a larger diameter. Live/dead assay further showed that ASCs within the spheroid were largely viable. Importantly, significant upregulation of stemness marker genes Sox-2, Oct-4 and Nanog was noted in cells within ASC spheroids comparing to monolayer culture. We also used western blot and immunofluorescence to confirm the enhanced expression of pluripotency markers in ASC spheroids. Secondly, we demonstrated a higher regenerative power of ASCs after spheroid formation by examining proliferation activity and colony-forming capability. Finally, we investigated the differentiation potentials of ASC spheroids when cultured in appropriate induction media. Not only differentiation capability toward adipogenic and osteogenic lineages (mesoderm) was maintained, but neurogenic (ectoderm) and hepatogenic (endoderm) differentiation of ASC spheroids was even enhanced after spheroid formation. To facilitate ASC transfer, we further designed a composite membrane made of chitosan/gelatin (C/G) for ASC culture and subsequent application to injured tissues. Increasing chitosan content within the C/G blends enhanced the sample’s mechanical properties, including tensile strength and elongation-at-break ratio. Although ASC spheroids developed shortly after seeding on pure chitosan films, increasing gelatin proportion in the C/G blends promoted cell adhesion onto the membranes. However, gradual gelatin release from the C/G blend films, leading to enriched chitosan content in the blends, encouraged ASC detachment and spheroid formation. We found that a blend made of 75% chitosan and 25% gelatin was most efficient in modulating ASCs for cell transfer. Therefore, C/G films in combination with ASCs have potentials of clinical application, such as treating a cutaneous wound. Healing of difficult cutaneous wounds is a challenging clinical problem, and current treatment modalities have their limitations. ASC can contribute to cutaneous wound healing through differentiation and paracrine effects. We observed that after spheroid formation of human ASCs on chitosan films, their stemness markers Sox-2, Oct-4 and Nanog were still maintained with spheroid dissociation and further monolayer culture. These spheroid-derived ASCs also expressed significantly more angiogenic growth factors, including hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), comparing to monolayer ASCs. The upregulation of CXCR4 in the spheroid-derived ASCs is associated with enhanced proliferation, reduced apoptosis and increased expression of matrix metalloproteinases. The interaction between stromal-derived factor-1 (SDF-1) and CXCR4 plays an important role in the homing of mesenchymal stem cells to the site of injury. In our animal study, we observed significantly more cellular engraftment of spheroid-derived ASCs in the cutaneous wound tissue with accelerated wound healing comparing to monolayer-cultured ASCs. The mechanism could be attributed to a higher cellular retention rate, enhanced differentiation and angiogenesis. Therefore, biomaterial modulation of ASCs for spheroid formation can provide important therapeutic potentials for tissue regeneration.