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標題: | 形成多細胞球體於間葉幹細胞分離與分化的應用 Formation of Multicellular Spheroids on the Selection and Differentiation of Mesenchymal Stem Cells |
作者: | Guo-Shiang Huang 黃國祥 |
指導教授: | 徐善慧 |
關鍵字: | 牙齦纖維細胞,間葉幹細胞,幾丁聚醣,透明質酸,三維多細胞球體,實體自由成型支架, Gingival fibroblasts (GF),mesenchymal stem cells (MSCs),chitosan, hyaluronan (HA),three-dimensional (3D) multicellular spheroids,solid freeform fabrication (SFF) scaffold, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | 本研究結合生醫材料與幹細胞發展一新穎的三維間葉幹細胞球體培養系統並探討此系統對於間葉幹細胞分離與分化的影響。在本研究的第一部分中,利用生醫薄膜發展一種新穎的幹細胞分離純化的方法。以兩種不同類型的牙齦纖維母細胞作為實驗模型,將這些牙齦纖維細胞培養在幾丁聚醣薄膜上24小時後,發現都可以分離出兩種不同群落的細胞,其中形成細胞球的群落”S”、沒有形成細胞球的群落”N”。所分離出的細胞球群落”S”會具有較高的幹細胞特性基因及神經脊基因表現且可以在長時間繼代培養後(大於10代)還維持群落形成能力(colony-forming ability)及細胞增生。同時也有更好的細胞分化能力。最後發現在幾丁聚醣薄膜上具有分離幹細胞的特性可能是藉由調控”S”群落的N-cadherin表現所造成。在第二部分的研究中,我們探討此促進間葉幹細胞(脂肪幹細胞及胎盤幹細胞)自我形成細胞球的生醫材料,對於維持幹細胞特性及促進軟骨分化的研究。脂肪幹細胞及胎盤幹細胞在本研究製備的幾丁聚醣或幾丁聚醣/透明質酸薄膜上培養數天,即會有細胞聚集成球的現象;且可維持幹性相關基因如Oct4, Sox2, and Nanog的表現。藉由CD44-blockage後會阻止成球的現象並使幹性基因表現下降。而經由Y-27632處理後會抑制細胞聚集形成細胞球,且幹性基因表現會大幅下降。而在幾丁聚醣/透明質酸薄膜上藉由生長因子進行軟骨分化七天後,發現軟骨分化之相關基因如Sox9、collagen type II (Col II) 及aggrecan的表現均增加,而組織學亦顯示軟骨樣細胞周圍有分泌豐富的軟骨細胞外基質。在第三部分的研究中,我們發展一種新穎的組織工程支架運用於承載3D多細胞球體。利用低溫冷凍擠壓成型法制備出實體自由成型(solid freeform fabricated)的聚乳酸聚-甘醇酸支架。經過大氣電漿處理在支架表面改質接枝上幾丁聚醣可以維持細胞球的型態。MSC細胞球種植SFF支架上,在in vitro及裸鼠實驗都可明顯觀察到糖胺多糖 及第二型膠原蛋白之分泌產生。本研究證實了三維間葉幹細胞球體培養系統可以有效從從異質細胞群中篩選出多分化潛能的幹細胞,且這種獨特的培養系統將有助於維持幹細胞的幹性和增加分化潛能,並可轉譯到三維支架上未來在再生醫學中有廣泛的應用。 A novel method was developed for isolating mulitpotent mesenchymal stem cells (MSCs) from human gingival fibroblasts (GF) by biomaterial membranes. Two typical but distinct types of GF, GF-A and GF-B, were grown from human gingival biopsies. They were characterized for surface markers by flow cytometry as well as the expressions of stemness and neural crest marker genes by RT-PCR. The two types of GF were slightly different in their surface markers; however, they had dramatic difference in the expression levels of stemness marker genes and neural crest marker genes. By culturing either type of GF on chitosan membranes for 24 hours, we were able to isolate two distinct subpopulations in each type of GF. We further characterized these cells, and determined the common properties shared by the spheroid forming subpopulation 'S', as well as those shared by the non-spheroid forming subpopulation 'N'. The subpopulation 'S' was capable of the multilineage differentiation, while the subpopulation 'N' was only efficient in osteogenic differentiation. GF-A and GF-B had different proportions of subpopulations. Chitosan as the cell culture substratum up-regulated the N-cadherin expression of the “S” but not “N” subpopulation, which may account for the cell sorting effect. This part of study showed that chitosan membranes could be used for isolation of the spheroid forming subpopulation in human GF that contained multipotent adult stem cells of which the number varied among donors and sites. Furthermore, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional (3D) multicellular spheroids that kept attached on the membranes. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. This part of study showed that formation of biomaterial-derived 3D MSC spheroids significantly promoted the chondrogenic differentiation. Finally, we revealed a novel design of next-generation scaffolds for delivering 3D cellular spheroids. Scaffolds with macroporosity to accommodate spheroids were made by solid freeform fabrication (SFF) from the solution of biodegradable poly(D,L-lactide-co-glycolide). In order to preserve the spheroid morphology, SFF scaffolds are surface-modified with chitosan following air plasma treatment. Human mesenchymal stem cell (MSC) spheroids loaded in SFF scaffolds produced a significantly larger amount of extracellular matrix in vitro and in NOD/SCID mice compared to single cells in the scaffolds. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15578 |
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顯示於系所單位: | 高分子科學與工程學研究所 |
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