臺灣博碩士論文加值系統

聽覺障礙(聽障)是最常見的聽力受損疾病，影響了各個年齡層的人們。每1000位新生兒就有2~3位患有聽障，嚴重可能導致耳聾。聽障是內耳毛細胞由於環境噪音、細胞老化、基因突變、耳毒性藥物、病毒感染所造成。在哺乳類動物中內耳毛細胞的失去是不可逆的，內耳毛細胞具有纖毛，其重要於聲音訊息的傳導。利用幹細胞進行毛細胞的分化已被證實具有可行性，如: 小鼠胚胎幹細胞、骨髓間葉幹細胞、誘導性多功能幹細胞和人類胚胎幹細胞等。肌陣攣癲癇症候群 (MERRF) 為粒線體疾病的一種，會引發癲癇、運動失調、視覺萎縮、聽覺障礙等病徵。大部分 MERRF 病人其病主要因為粒線體 DNA 在第8344個核苷酸產生由腺嘌呤(Adenine)變成鳥糞嘌呤 (Guanine) 的突變，導致胺基酸轉移核苷酸(tRNALys)的功能異常。在本研究中，我們首先將誘導性多功能幹細胞誘導分化成內耳毛細胞，並建立人類誘導性多功能幹細胞之內耳毛細胞分化的平台。我們利用此平台去誘導帶有A8344G粒線體DNA突變的誘導多功能幹細胞分化成內耳毛細胞並利用此內耳毛細胞分化平台去篩選能促進內耳毛細胞分化的基因。結果顯示，使用人類多功能幹細胞所分化出來的類毛細胞具有經典毛細胞的基因表達及蛋白質表現。然而，利用電子掃描顯微鏡觀察時，可以發現到類毛細胞的細胞表面呈現未成熟的毛束構造。帶有A8344G粒線體突變的人類誘導多功能幹細胞亦可進行內耳毛細胞的誘導分化，但所分化之纖毛則無複雜之立體結構。更進一步地，在帶有A8344G 粒線體DNA突變的人類誘導多功能幹細胞和經分化而得的類毛細胞中，我們發現到高反應性的活性氧分子(ROS)的累積及抗氧化基因表現的上升。此外，我們發現一個對於纖毛形成重要的轉錄因子X (TF-X)，其對人類誘導性多功能幹細胞之內耳毛細胞分化可能具有調控的效果，實驗結果顯示 TF-X單獨存在並不會對內耳毛細胞之分化有顯著的影響，但是TF-X和已知的內耳纖毛增生轉錄因子ATOH1同時作用下可以觀察毛細胞基因的表現顯著上升及獲得結構上更加成熟的纖毛束。由以上結果顯示A8344G粒線體DNA突變會促使高反應性的活性氧分子增加，影響抗氧化基因的表現。TF-X和ATOH1基因共同表現能促使內耳毛細胞的毛束構造更加成熟。本研究在再生醫學上將有助於內耳毛細胞再生的發展，並在臨床應用上有助於聽覺障礙的治療方式突破以及疾病機轉的探討。

Hearing impairment (HI) is the most common hearing-loss disorder, it affects peoples of all ages. Approximately 2~3 in 1,000 children are born with impaired hearing, seriously it may cause deafness. HI is caused by environmental noise, aging, genetic mutation, ototoxic drugs or viral infections. In mammals, losing of inner ear hair cells (HCs) is irreversible. HCs differentiation is feasible and has been demonstrated in different types of stem cells, such as bone marrow-derived mesenchymal stem cells, mouse and human embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs). Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disease characterized by myoclonus epilepsy, ataxia, ragged-red fibers, myopathy and sensorineural hearing loss. MERRF syndrome is primarily caused by an A to G point mutation at mitochondrial DNA (mtDNA) 8344 that disrupts tRNALys and causes protein synthesis dysfunction. In this study, we differentiated human induced pluripotent stem cells (hiPSCs) into HCs and generated HCs that carry the mtDNA A8344G mutation from MERRF-hiPSC. In addition, we identified a transcription factor X (TF-X) that is crucial for ciliogenesis. Our results showed that hiPSC derived from MERRF patients (MERRF-hiPSCs, harboring A8344G mutation) had similar otic differentiation ability to hiPSC without mtDNA A8344G mutation. MERRF-hiPSC-derived HCs could express classical hair cell (HC) markers such as Myosin 7A and Espin, but failed to exhibit stereociliary bundles. MERRF-hiPSC and MERRF-hiPSC-derived HC-like cells exhibited significantly higher reactive oxygen species (ROS) production and upregulated antioxidant genes expression. Furthermore, we demonstrated that TF-X combination with ATOH1 could significantly upregulate HC marker genes expression and promote the differentiated HCs with more mature stereociliary bundles on cell surface. In conclusion, these findings showed that MERRF-hiPSCs and –HCs carrying the mtDNA A8344G mutation displayed contained accumulated ROS levels, upregulated antioxidant genes expression and hair bundles with abnormal structure. TF-X combination with ATOH1 promoted the HCs with more mature stereociliary bundles. The gained knowledge from this study will facilitate the development of HCs regeneration for regenerative medicine, and generation of patient-specific HCs for disease modeling of deafness.

中文摘要 I
Abstract III
Table of Contents V
List of Figures VII
List of Tables VIII
Abbreviation IX
Chapter 1 Introduction 1
1-1. Hearing loss 2
1-2. Auditory system in humans 3
1-3. Inner ear HCs 4
1-4. Stem cells 5
1-5. ESCs and iPSCs 6
1-6. Regeneration of HCs from stem cells 7
1-7. Role of Atoh1 transcription factor in inner ear HCs development 8
1-8. Mitochondria 9
1-9. Mitochondrial DNA mutation and reactive oxygen species 11
1-10. Mitochondrial disease : myoclonic epilepsy associated with ragged-red fibers syndrome (MERRF) 12
1-11. Significance 13
Chapter 2 Materials and Methods 14
2-1. Cells 15
2-2. Embryoid body formation 15
2-3. Otic progenitors induction 15
2-4. Inner ear HCs differentiation 16
2-5. Reverse transcription polymerase chain reaction (RT-PCR) 16
2-6. Quantitative polymerase chain reaction (qPCR) 16
2-7. Immunocytochemistry (ICC) 16
2-8. Scanning electron microscopy (SEM) 17
2-9. Flow cytometry 17
2-10. mtDNA A8344G mutation sequencing 17
Chapter 3 Results 19
3-1. Generation of inner ear HC-like cells from hiPSCs 20
3-2. Specific gene marker expression during HC differentiation derived from hiPSCs 21
3-3. The expression of HC markers in HC-like cells after long-term differentiation 22
3-4. Mechanosensory hair cell-like cells fail to acquire mature stereociliary bundles 23
3-5. mtDNA A8344G mutation do not interfere otic differentiation process 23
3-6. mtDNA A8344G mutation causes ROS overproduction and alternates antioxidant genes expression in hiPSCs and hiPSC-derived inner ear HCs 25
3-7. The ATOH1 and TF-X gene combination promotes inner ear HC differentiation and the formation of stereociliary bundles 26
Chapter 4 Discussion 28
4-1. Generation of human HCs from hiPSCs in vitro 29
4-2. Diseases-specific hiPSC as a disease model 29
4-3. MtDNA A8344G leads to mitochondrial dysfunction 30
4-4. HCs with MYO7A mutation falls to acquire stereociliary bundles. 31
4-5. Conclusion 32
Reference 33

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