在2D及3D環境培養人類脂肪幹細胞其多能性及分化能力

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研究生：	謝肇文 Chao-Wen Hsieh
論文名稱：	在2D及3D環境培養人類脂肪幹細胞其多能性及分化能力 Pluripotency and Differentiation Ability of Human Adipose-Derived Stem Cells Culture in 2-D and 3-D culture
指導教授：	樋口亞紺 Akon Higuchi
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	83
中文關鍵詞：	人類脂肪幹細胞、2D培養、3D培養、多能性、分化能力
外文關鍵詞：	human Adipose-derived Stem cells, 2D cultivation system, 3D cultivation system, Pluripotency, Differentiation Ability
相關次數：	點閱：10 下載：0
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充間質幹細胞於再生醫學領域中被普遍認為是十分具有潛力的細胞來源相較於胚胎幹細胞以及誘導多能性幹細胞。這是因為充間質幹細胞不會如胚胎幹細胞產生道德方面的疑慮以及誘導多能性幹細胞在體內會有產生腫瘤發生的可能。但應用上的問題則在於充間質幹細胞會有無法長久培養及多能性遠遠低於胚胎幹細胞和誘導多能性幹細胞。
在之前的研究發現，人類脂肪幹細胞在TCPS培養盤上，其多能性基因 (Oct4,Sox2,Nanog)會急速的下降。但相較於含有許多細胞自脂肪中所萃取出的SVF溶液以及在培養液中懸浮培養圓球體符合幹細胞在胚胎中最原始的型態，培養在TCPS培養盤上面的多能性則遠遠不足。
在此研究中，我們在TCPS培養盤以及Ultra low attachment培養盤上面培養人類脂肪幹細胞。幹細胞會貼附在TCPS培養盤上並開始生長於表面，稱之為2D培養。則細胞培養在Ultra low attachment培養盤中，人類脂肪幹細胞會懸浮於培養液中所以稱為3D培養。在一段時間的培養後，我們會因為其培養環境不同去比較其多能性以及分化人類脂肪幹細胞為成骨細胞，軟骨細胞去比較培養環境的不同其分化能力。並透過免疫螢光染色法以及多能性基因的檢測去比較兩種培養方式的多能性。
結果顯示在懸浮培養中，其多能性都會優於培養在TCPS培養盤上的幹細胞。甚至於，培養在較低多能性的TCPS培養盤上後，幹細胞接著被培養在懸浮培養環境中，我們發現其多能性會回復以及維持較高多能性。分化能力亦然，不論是成骨分化或者軟骨分化，懸浮培養出的幹細胞，其分化能力都優於貼附培養後的幹細胞。

Human adult stem cells, such as human adipose-derived stem cells, are considered to be an attractive source of stem cells than human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This is because human adult stem cells do not generate the ethical concerns that accompany in hESCs. Although human adipose-derived stem cells (hADSCs) are promising for use in regenerative medicine, their lower expansion ability (aging problem) due to the lower pluripotency of hADSCs compared with hESCs and hiPSCs is a critical issue.
It’s found that the pluripotency gene expression of Oct4, Sox2, and Nanog in hADSCs after cultivation on TCPS dramatically decreased compared to those in the cells in stromal vascular fraction (SVF) as well as the cells in 3-D culture. There are high pluripotent stem cells in SVFs and 3-D culture, although SVF has more heterogeneous population compared to hADSCs cultured on tissue culture polystyrene (TCPS) dishes.
It’s evaluated whether hADSCs can be explained by “Stochastic model” or “Elite model” by hADSCs culture in 2-D culture and 3-D culture sequentially. Precisely, we evaluated the difference of gene expression of the cells when hADSCs were cultured in the 2-D condition (TCPS) and in 3-D condition (ultra low attachment dish) sequentially. It is found that the pluripotent gene expression of hADSCs (Oct4, Sox2 and Nanog) in suspension (3-D culture) is higher than that on TCPS (2-D culture). Especially, low pluripotent gene expression of hADSCs cultured on TCPS is changed to be higher pluripotent gene expression after hADSCs are shifted in 3-D culture

Abstract    I
摘要    II
Index of figures    VI
Index of Tables    VIII
Chapter 1 Introduction    1
1.1    Stem Cells    1
1-1-1  Embryonic Stem Cells (ESCs)    2
1-1-2  Induced pluripotent stem cells (iPSCs)    2
1-1-3  Mesenchymal stem cells (MSCs)    3
1-2 Human adipose-derived stem cells (hADSCs)    4
1-2-1 Isolation of human adipose-derived stem cells    5
1-2-2 Membrane filtration method to purify hADSCs.    6
1-2-3 Membrane migration method to culture hADSCS    6
1-2-4 Differentiation capacity of human adipose-derived stem cell for clinical application    7
1-3 Three dimensional spheroid cell culture of human ADSCs.    9
1-3-1 Preparation for Spheroid Formation    10
1-4 Characterization of human ADSCs    13
1-4-1. Flow-cytomertry    13
1-4-2. Quantitative real time polymerase chain reaction    14
1-4-3. Immunofluorescence staining    16
Chapter 2 Materials and Methods    17
2-1 Materials    17
2-1-1 Sources for adipose tissue    17
2-1-2 Commercial culture dishes    17
2-1-3 Culture medium/Differentiation induction medium    17
2-1-4 Phosphate buffer saline solution (PBS)    17
2-1-5 Digestion solution    17
2-1-6 ACK lysing solution    18
2-1-7 Flow cytometry    18
2-1-8 Immunostaining    18
2-1-9 RNA extraction kit    18
2-1-10 Reverse transcription kit    18
2-1-11 Real-time polymerization chain reaction    19
2-1-12 qRT-PCR probe    19
2-1-13 Alkaline phosphate assay    19
2-1-14 Alizarin Red S staining    19
2-1-15 von Kossa staining    19
2-1-16 Alcian blue staining    19
2-2 Experiment method    20
2-2-1Preparation of culture medium    20
2-2-2 Isolation and culture of adipose-derived stem cells    20
2-2-3 Culture and passage of hADSCs    22
2-2-4 Cell density measurement    23
2-2-5 Isolation of total RNA    23
2-2-6 Reverse transcription of mRNA into DNA    24
2-2-7 Quantitative real time polymerase chain reaction    25
2-2-8 Differentiation of adipose-derived stem cells    25
2-2-9 Alkaline phosphatase activity    26
2-2-10 Alizarin Red S staining    26
2-2-11 von Kossa staining    27
2-2-12 Alcian blue staining    27
2-2-13 Quantitative analysis of differentiation    28

Chapter 3 Results and Discussion    29
3-1 Cultivation of hADSCs in 2D and 3D culture    29
3-2 Pluripotency analysis of hADSCs cultured in 2-D and 3-D culture    32
3-3. Differentiation ability of hADSCs into osteogenic differentiation, which were cultured in 2-D and 3-D culture in advance    40
3-4. Differentiation ability of hADSCs into chondrogenic differentiation, which were cultured in 2-D and 3-D culture in advance    46
Chapter 4 Conclusion    48
References    50
Appendix data    58

                                

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