人類多能幹細胞分化視網膜色素上皮細胞培養於各種塗佈細胞外間質

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研究生：	朱昱如 Yu-Ru Zhu
論文名稱：	人類多能幹細胞分化視網膜色素上皮細胞培養於各種塗佈細胞外間質 Differentiation of Human Pluripotent Stem Cells into Retinal Pigmented Epithelium on ECM-coated Surfaces
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：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	102
中文關鍵詞：	細胞外基質、生醫材料、人類多能幹細胞、細胞分化、視網膜色素上皮細胞
外文關鍵詞：	ECM, biomaterials, human pluripotent stem cells, cell differentiation, retinal pigment epithelium
相關次數：	點閱：11 下載：0
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人類多能幹細胞包括人類胚胎幹細胞和人類誘導多能幹細胞，而人類多能幹細胞可成為用於特定疾病的潛在療法。來自世界衛生組織的資料顯示老年性黃斑部病變是視力損害的第三大原因，且預計未來會變得越來越嚴重。80-90% 的老年性黃斑部病變主要由視網膜色素上皮的功能損壞而引起。幸運的是，移植人類多能幹細胞分化成的視網膜色素上皮細胞可以作為治癒老年性黃斑部病變疾病的方法。然而，來自人類多能幹細胞的視網膜色素上皮通常存在著純度不足和培養時間長的問題。因此，在本篇研究中有比較不同的培養製程和嘗試各種細胞外基質來評估哪些條件最適合人類多能幹細胞分化為視網膜色素上皮細胞。我測試了人類多能幹細胞分化為視網膜色素上皮細胞三種類型的培養製程（N2、NIC84 和Activin A 培養製程）。每個培養製程都經過進一步修改來提高人類多能幹細胞分化為成熟視網膜色素上皮的效率。在本研究中，我以原來的NIC84培養製成進行以下調整，(1) 降低 CTM 濃度；(2) 改變細胞繼代方式；(3) 減少製成前28天的培養液使用量。經過這些調整，可以用此改良後的培養製成高效地獲得從人類誘導多能幹細胞分化成的成熟視網膜色素上皮細胞。在此研究中，也有對這些細胞進行相關的驗證程序及檢測。視網膜色素上皮細胞因為有色素沉澱之功能，呈色為棕色，而我從人類誘導多能幹細胞誘導成的視網膜色素上皮細胞也呈棕色。加上這些視網膜色素上皮細胞有表達ZO-1和 RPE65 的這兩個代表成熟視網膜色素上皮細胞的標記。透過細胞顏色的轉變及特定抗體的標記，可證明改良後的NIC84培養製程可使人類多能幹細胞分化成視網膜色素上皮細胞。接著在不同的細胞外基質上，透過特定抗體的標記比例及細胞生長速率我發現擁有Matrigel 塗層和 LN-521 塗層之培養皿較擁有rVN 塗層、LN-511 塗層和 Synthemax II 塗層之培養皿更能支持人類多能幹細胞分化成視網膜色素上皮細胞。

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), where hPSCs can be used for the potential cell therapy toward specific disease. Age-related macular degeneration (AMD), which ranks the third leading cause of vision impairment according to world health organization (WHO), is expected to become more and more serious in the future. 80-90% of AMD disease mainly results from the dysfunction of the retinal pigmented epithelium (RPE). Fortunately, transplantation of hPSCs-derived RPE can serve as a regenerative approach to cure AMD disease. However, RPE derived from hPSCs usually suffers from insufficient purity and long culture period. Therefore, different protocols and cell culture extracellular matrices (ECMs) were investigated to evaluate which conditions would be the most suitable for hPSCs to differentiate into RPE in this study. Three types of protocols (N2, NIC84, and Activin A protocols) for hiPSC differentiation into RPE were evaluated. Each protocol was further modified to improve the efficiency of differentiation into mature RPE. Mature RPE differentiated from hiPSCs can be obtained with high efficiency using the modified protocol of NIC84 with reducing concentration of CTM and modified medium in day 28-30 of differentiation, which was developed in this study. hiPSC-derived RPE showed brown color indicating pigmented cells (mature RPE) and expressed mature RPE marker of ZO-1 and RPE65. Matrigel-coated and LN-521-coated dishes supported high differentiation of hiPSCs into RPE compared to rVN-coated, LN-511-coated, and Synthemax II-coated dishes.

Abstract    I
Index of content    III
Index of Figure    VI
Index of table    XI
Chapter 1. Introduction    1
1-1.    Stem cells    1
1-1-1. Embryonic stem cells    2
1-1-2. Induced pluripotent stem cells    4
1-1-3. The limitations of human embryonic stem cells and induced pluripotent stem cells    5
1-1-4. Stem cells for therapeutic application in future    6
1-2.    Human Pluripotent Stem Cells Cultivation    10
1-2-1. Pluripotent maintenance of hPSCs maintenance on feeder cell layers    11
1-2-2. Feeder-free and xeno-contained substrates for hPSCs cultivation    11
1-2-3. Human PSC culture on feeder-free and xeno-free extracellular matrices    12
1-3.    Differentiation of human Pluripotent Stem Cells (hPSCs) into retinal pigmented epithelium (RPE)    15
1-3-1. Effective protocols for preparation of hPSCs-derived RPE    16
1-3-2. Preparation of hPSCs-derived RPE by spontaneous differentiated method    17
1-3-3. Preparation of hPSCs-derived RPE by small-molecule usage method    18
1-3-4. Preparation of hPSCs-derived RPE by neural differentiation factors usage method    19
1-3-5. hPSCs-derived RPE preparation in xeno-free and feeder-free conditions and automated systems    19
1-4.    Characterization of retinal pigmented epithelium (RPE) derived from hPSCs    20
1-4-1. Morphology of hPSCs-derived retinal pigmented epithelium (RPE)    20
1-4-2. Flow cytometry analysis of retinal pigmented epithelium (RPE) derived from hPSCs    21
1-4-3. Immunofluorescence staining analysis of retinal pigmented epithelium (RPE) derived from hPSCs    23
1-5.    The goal of this study    24
Chapter 2. Materials and Methods    25
2-1. Materials    25
2-1-1. Cell Line    25
2-1-2. Commercial Culture Dishes    25
2-1-3. Commercial Coated Substrates    25
2-1-4. Medium and Other    25
2-1-5. Chemicals for immunostaining    27
2-1-6. Chemicals for flow cytometry    28
2-2. Methods    28
2-2-1. Preparation of ECM protein-coated dishes    28
2-2-2. Human pluripotent stem cells cultivation and passage    29
2-2-3. Seeding density measurements of hPSCs    30
2-3. Retinal pigmented epithelium (RPE) differentiation    30
2-3-1. Preparation of differentiation medium    30
2-3-2.    Differentiation protocols of hPSCs into retinal pigmented epithelium    32
2-4. Characterization of hPSC-derived RPE    37
2-4-1. Immunofluorescence staining of hPSC-derived RPE    37
2-4-2. Flow cytometry measurements of hPSC-derived RPE    39
Chapter 3. Results and Discussion    42
3-1. Preparation of hiPSC-derived RPE using N2 protocol    42
3-1-1. Morphology of hiPSCs-derived RPE on different ECM protein-coated dishes using N2 protocol    42
3-1-2. Immunostaining of hiPSCs-derived RPE differentiated using N2 protocol    46
3-1-3. LN511 was the most suitable coating subtrate using N2 protocol but N2 protocol could not generate hiPSC-derived RPE efficiently    47
3-2. Preparation of hiPSC-derived RPE using NIC84 Protocol    47
3-2-1. Modification of NIC 84 protocol    47
3-2-2. Morphology of hiPSCs-derived RPE on different ECM protein-coated dishes using final version of modified NIC 84 protocol    51
3-2-3. Cell proliferation rate of hiPSCs-derived RPE on different ECM protein-coated dishes using final version of modified NIC 84 protocol    55
3-2-4. Immunostaining of hiPSCs-derived RPE on different ECM protein-coated dishes using final version of modified NIC 84 protocol    57
3-2-5. RPE marker expression of hiPSCs-derived RPE on different ECM protein-coated dishes using final version of modified NIC 84 protocol by flow cytometry analysis    61
3-2-6. Matrigel- and LN521-coated dishes were the most suitable cell culture biomaterials in final version of modified NIC84 protocol, which could generation RPE efficiently    63
3-3. Preparation of hiPSC-derived RPE using Activin A protocol    64
3-3-1. Morphology of hiPSCs-derived RPE on different ECM protein-coated dishes using Activin A protocol    64
3-3-2. Flow cytometry analysis of hiPSCs-derived RPE using Activin A protocol    65
3-3-3. LN511 was the most suitable substrates for hiPSC-derived RPE using Activin A protocol with high RPE generation efficiently    66
Chapter 4. Conclusion    67
Reference    69
Appendix    76
 

                                

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