摘要:隨著幹細胞研究的快速進展,幹細胞療法已成為具有潛力的生醫學療法。脂肪幹細胞(adipose-derived stem cell, ASC)因來源豐富及容易取得,已成為考慮幹細胞治療的極佳細胞來源。然而,移植單顆、分散的脂肪幹細胞到受傷組織卻常發現產生早期的細胞死亡,同時治療效果十分有限。相較於分散的脂肪幹細胞,利用細胞薄片 (cell sheet)之技術應有益於保留細胞連接(cellular junctions)及內生的細胞外基質(endogenous extracellular matrix, ECM),進而可促進移植細胞之存活。然而在文獻中尚未對以脂肪幹細胞製備細胞薄片之技術有完整的探討,所以本研究的目標是探索將脂肪幹細胞薄片應用於組織再生的可能性。在早期的研究中,我們已成功地使用維生素C 刺激脂肪幹細胞分泌細胞外基質,而可成功製備細胞薄片。我們還有一重要發現,即細胞薄片內的脂肪幹細胞幹性標記 (stemness markers) , 包含Oct4、Sox2 、Nanog 的基因及蛋白質表現皆顯著增強。因此,我們在這項研究提出的中心假說為:由脂肪幹細胞製備的細胞薄片可以表現出優異的生物特性,進而可促進組織再生。首先,我們將探討維生素C 對脂肪幹細胞增生、衰老、幹性 (stemness)及分化的影響,並了解其機轉。再者,我們將利用幾丁質 (chitosan)和明膠 (gelatin)溶解特性的不同,而發展出一個混合幾丁質和明膠的支架促進脂肪幹細胞薄片的形成及轉移。我們也希望能將脂肪幹細胞薄片進行去細胞化 (decellularization),製造出一種以細胞外基質為基底的新生醫材料,以應用於促進組織再生。此外,我們將建立糖尿病小鼠的皮膚傷口癒合的動物模式,並利用此模式來測試脂肪幹細胞薄片或已去細胞化的脂肪幹細胞薄片在生物體內是否具增強組織再生的能力。概括來說,本研究具有以下4 個特定目標:目標#1:我們將使用一種較穩定的維生素C(Ascorbate-2-phosphate, A-2-P)來促進脂肪幹細胞生成細胞外基質,以製備細胞薄片。我們假設A-2-P 的作用可以提高脂肪幹細胞的再生性能,我們並將進一步研究A-2-P 針對脂肪幹細胞增殖,衰老,幹性,和分化能力的影響。目標#2:製備經交聯的幾丁質/明膠薄膜,使脂肪幹細胞可循序在其上形成細胞薄片和轉移到損傷部位。我們假設幾丁質/明膠薄膜中之明膠降解速度較快,明膠逐步釋放後複合膜中幾丁質所占比例提高,造成細胞較不易貼附,逐步使細胞薄片脫離複合膜而與受傷的組織黏著。目標#3:製備去細胞化的脂肪幹細胞薄片以應用於促進組織再生。去細胞化的脂肪幹細胞薄片不具有免疫排斥問題,可使用於異體組織。此外,去細胞化的脂肪幹細胞薄片亦可結合新鮮分離的脂肪間質血管部分 (stromal vascular fraction, SVF)細胞以應用於組織再生 ,可以規避在體外培養病患自身脂肪幹細胞以形成細胞薄片的步驟,從而導致更快的臨床應用。目標#4:糖尿病傷口癒合是一個困難且具挑戰性的臨床問題,亦提供一個很好的模型來研究幹細胞的再生能力。我們將以糖尿病小鼠的皮膚傷口癒合為動物模式,測試幾丁質/明膠薄膜結合脂肪幹細胞薄片,觀察是否可以有效地促進糖尿病小鼠之傷口癒合。我們亦會測試去細胞化的脂肪幹細胞薄片結合脂肪間質血管部分細胞,觀察其對糖尿病小鼠傷口癒合之影響。本計劃擬去探索及利用脂肪幹細胞的內在能力去建構組織基質,以進一步應用於再生醫學。在完成這個計畫時,我們預期建立一個可行的流程來製造脂肪幹細胞薄片,並隨後能藉由幾丁質/明膠薄膜之特性,順利將細胞薄片轉移到受傷的組織以促進組織再生。我們也預期能開發由脂肪幹細胞薄片的細胞外基質衍生而來的新型生醫材料,將來可應用於組織再生。脂肪幹細胞薄片及衍生的細胞外基質材料將會進一步以動物實驗測試其療效,我們認為這項細胞薄片技術很有潛力能廣泛應用於脂肪幹細胞相關的細胞療法。
Abstract: With rapid progress in stem cell research, stem cell therapies have become promising therapeutic approaches for regenerative medicine. The abundance and easy accessibility of adipose-derived stem cell (ASC) have made it a promising candidate for stem cell therapies. However, transplantation of dissociated ASCs is frequently associated with early cell death with limited therapeutic effects. It has been proposed that the use of cell sheets is beneficial for cell transplantation by preserving cellular junctions and endogenous extracellular matrix. However, cell sheet fabrication with ASCs has not been thoroughly investigated in the literature, so we aim to explore the potential applications of ASC sheets in tissue regeneration.In our preliminary study, we have successfully stimulated extracellular matrix (ECM) secretion of ASCs and fabricated cell sheets by vitamin C treatment. Interestingly, we also found enhanced expression of stemness markers, including Oct4, Sox2 and Nanog, in ASCs within cell sheets. Therefore, we propose in this study a central hypothesis that ASCs within cell sheets exhibit superior biological properties of regenerative capabilities.In this study, we will first investigate the effect of vitamin C on the proliferation, senescence, stemness, and differentiation capabilities of ASCs. Taking advantage of the different dissolution properties of chitosan and gelatin, we will next develop composite chitosan/gelatin films to facilitate the formation and transfer of ASC sheets. We also aim to decellularize ASC sheets to fabricate a novel ECM-based biomaterial for tissue regeneration. Moreover, the capability of the original or decellularized ASC sheet to enhance tissue regeneration will be tested in a diabetic murine model of impaired cutaneous wound healing. In summary, we will pursue the following four specific aims:Aim #1: To enhance cell sheet formation by treating ASCs with a stable form of vitamin C, ascorbate-2-phosphate (A-2-P). The effect of A-2-P on the proliferation, senescence, stemness, and differentiation capabilities of ASCs will be investigated.Aim #2: To develop a crosslinked chitosan/gelatin film to facilitate the sequential formation and transfer of ASC sheets. After ASC attachment and sheet formation on the composite film, the cell sheet-coupled film can be applied to the injury site to allow gradual cell sheet detachment and integration of the sheet into the injured tissue.Aim #3: To decellularize the ASC sheet for fabrication of a novel biomaterial for tissue regeneration. ECM derived from ASC sheets alone allows allogeneic application. Moreover, its combination with freshly isolated adipose stromal vascular fraction (SVF) cells may circumvent in vitro culture of patient’s autologous ASCs for sheet formation, leading to faster clinical translation in regenerative medicine.Aim #4: Diabetic wound healing is a challenging clinical issue and represents a good model to investigate the regenerative capabilities of ASC sheets. The chitosan/gelatin film-coupled ASCsheets will be tested in a diabetic murine model of impaired cutaneous wound healing.Decellularized ASC sheets in combination with SVF cells will also be investigated in the same model for enhancing diabetic wound healing. The proposed work is designed to explore and utilize the inherent capabilities of ASCs to engineer tissue constructs for regenerative medicine. At the completion of this project, we expect to establish a feasible biomaterial-assisted protocol of ASC sheet fabrication and subsequent transfer to the injured tissues. We also expect to develop a novel ECM-based biomaterial derived from ASC sheets for potential application in tissue regeneration. The therapeutic efficacy of ASC sheets and sheet-derived ECM will be further tested in an animal model of diabetic wound healing. We believe that cell sheet technology exhibits great potentials in applying ASCs for a broad range of cell-based therapeutics.