Development of cationic nanoparticles with Sox2, Klf4, Oct4 and c-Myc proteins in induced pluripotent stem cells’ generation

人類誘導多能性幹細胞 (induced pluripotent stem cells, iPS cells) 是一種將體細胞再程序化後產生的人造幹細胞，其特點在於將已分化的細胞退分化回具有分化能力的幹細胞，改變了過往認為分化只能是單行道的概念。目前最主要的人類誘導多能性幹細胞製造方法是山中伸彌博士於 2006 年所發展，利用帶有四因子 (Klf4, Sox2, Oct4 以及 c-Myc) 的反轉錄病毒感染體細胞再程序化而成。自該研究之後，許多藉由 DNA 嵌入宿主基因體來表現四因子，進一步將體細胞再程序化成為人類誘導多能性幹細胞的方式陸陸續續被發展出來，這些以 DNA 作為因子運送載體的人類誘導多能性幹細胞也被稱為第一代人類誘導多能性幹細胞。 然而，雖然以 DNA 做為四因子運送載體其再程序化的效率比其他方法來的高，此種方法的最大缺點在於載體所攜帶的四因子是隨機嵌入細胞染色體中，宿主基因突變的疑慮導致了第一代人類誘導多能性幹細胞在醫療上的發展大受限制。為了解決基因嵌入的問題，利用非 DNA 的載體來製造人類誘導多能性幹細胞成為近期研究重心。這些非 DNA 的載體包括蛋白質、信使 RNA 以及微小 RNA (MicroRNA) 等，其共通特點在於再程序化的過程中不需對宿主基因體做任何更動，其誘導出來的人類誘導多能性幹細胞具有更佳的醫療應用前景。 基於前述理由，在此研究中我希望利用蛋白質作為四因子的載體送入人類包皮纖維母細胞 (HS68) 中來誘導人類誘導多能性幹細胞的產生。蛋白質具有易於大量表現、純化以及儲存的優點，其作為載體的主要障礙在於蛋白質無法主動穿過細胞膜，需藉由奈米粒子或穿膜短肽的幫助才能順利進入細胞內；因此，我利用醫工所開發的 gelatin-polyethyleneimaine (gelatin-PEI) 奈米粒子來包裹綠色螢光蛋白 (作為模式蛋白) 以及四因子,藉此將這些蛋白質送入人類纖維母細胞中，並在吞吃後藉由奈米粒子上的一級氨與二級氨作為質子海綿 (proton sponge) 協助蛋白質逃脫核內體 (endosome)。 在奈米粒子的吞吃效率測試中，可見當濃度提升到 50ug/ml 時，HS68 能夠吞吃將近九成的奈米粒子；而在細胞毒性測試中，HS68 雖會在投藥後減少約三成的細胞，卻能在三天的培養後回復到原有數量，顯示未來再程序化時反覆投藥是可行的。 而在蛋白質表現的結果中可見，不論是四因子或是綠色螢光蛋白都能成功的在大腸桿菌表現系統中表現，同時此五種蛋白質也都能利用鎳親合管柱來進行純化。在純化後的綠色螢光蛋白接受 gelatin-PEI 奈米粒子包裹後，我將兩者的混合物加入 HS68 的培養基中，發現綠色螢光蛋白以及奈米粒子能順利被細胞吞吃，且綠色螢光蛋白依舊能產生螢光。顯示利用這樣的奈米粒子去包裹蛋白質來送入細胞是可行的，對於蛋白質的損傷也是可接受的。 以上研究結果顯示未來利用 gelatin-PEI 奈米粒子來包裹四因子蛋白，藉此進行 體細胞再程序化製造人類誘導多能性幹細胞是值得嘗試的,未來這樣的方法可能成為更安全的細胞再程序化方式。Induced pluripotent stem cells (iPS cells) are artificial stem cells, generated by somatic cell reprogramming. In 2006, Shinya Yamanaka and Kazutoshi Takahashi investigated 24 candidates that are specifically expressed in embryonic stem cells (ES cells) as pluripotent-correlated genes, they finally found out that Klf4, Sox2, Oct4 and c-Myc, which are known as Yamanaka factors, are able to derive iPS cells from adult fibroblasts. Since then many DNA-dependent reprogramming methods have been developed, and these methods have the same problem, which hinder the clinical application of this type of iPS cells. The problem of DNA-dependent methods is uncontrollable genome integration during reprogramming process, so the following researcher focused on DNA-free reprogramming vectors, such as proteins, microRNA and mRNA. Those DNA-free methods won’t modify host genome and therefore those methods are more promising in regenerative medicine area. Based on these reasons, I used proteins as DNA-free reprogramming vectors to generate iPS cells. Proteins are easy to overexpress, purify and store up, but without specific peptide sequence or protein carrier, most proteins are unable to cross cell membrane, and hence I cooperated with Dr. Yi-You Huang and Dr. Ming-Ju Chou using their gelatin–polyethyleneimine (gelatin-PEI) nanoparticles as my protein carrier. On the other hand, gelatin-PEI nanoparticles have many primary and secondary amines work as proton sponge, which provide high efficiency of endosomal escape. In my research, the uptake efficiency experiment performed by flow cytometry showed that HS68 cells are able to uptake almost 90% of gelatin-PEI nanoparticle when particle concentration reach 50ug/ml. Besides, cell viability assay showed that although HS68 population will decrease after 24 hours particle application, HS68 cells will repopulate after three days, indicating that repeating protein delivery is possible. On the other hand, I also showed that both enhanced green fluorescent proteins (eGFPs) and Yamanaka four factors can be overexpressed in Escherichia coli expression system, and these proteins were able to be purified by Ni-NTA affinity columns. Furthermore, after I mixed gelatin-PEI nanoparticles and eGFPs with HS68 cells, these particles are able to transport our model protein eGFPs into HS68 cell line, and transfection process won’t cause severe cell death. Overall, these data showed that gelatin-PEI nanoparticles are able to carry our model proteins, eGFPs, into human foreskin fibroblasts. I suppose that we can combine proteins and nanoparticles as reprogramming vectors, and this might be a new method to generate iPS cells口試委員會審定書 i Acknowledgement ii 中文摘要 iii Abstract v Table of contents vii List of figures ix List of tables x Chapter one Introduction 1 1.1 Induced pluripotent stem cells: History, characteristics and further applications 1 1.2 Factor delivery: First step in iPS cells generation 4 1.3 Yamanaka factors: Sox2, Klf4, Oct4 and c-Myc 6 1.5 Gelatin- Polyethyleneimaine (Gelatin-PEI) nanoparticles 8 1.6 Research aims 11 Chapter two Materials and methods 13 2.1 Cell culture 13 2.1.1 Cell line 13 2.1.2 Cell culture condition 13 2.2 Plasmid construction 13 2.2.1 Plasmid 13 2.2.2 Polymerase chain reaction (PCR) condition 14 2.2.3 Gel filtration 14 2.2.4 Restriction enzyme digestion 15 2.2.5 Competent cell preparation 15 2.2.6 Ligation and transformation 16 2.3 Plasmid mini-preparation 17 2.4 Plasmid midi preparation 17 2.5 Total protein extraction. 18 2.6 SDS-PAGE and CBR staining. 19 2.7 Western analysis 20 2.8 Affinity chromatography. 21 2.9 Protein concentration determination 22 2.10 Cell viability analysis 22 2.11 Cellular uptake analysis 23 2.10 Intracellular protein delivery of gelatin-PEI nanoparticles 24 Chapter three Result 26 3.1 Identification of His-eGFP and His-Yamanaka factors overexpressed by BL21 and Rosetta™ competent cells 26 3.1.1 Total protein analysis by coomassie brilliant blue and Western blotting 26 3.1.2 Induction time optimization of His-eGFP and His-SKOM 27 3.2 Purification of His-eGFP and His-Yamanaka factors by affinity columns 28 3.3 Cellular uptake of gelatin-PEI nanoparticles in HS68 cell line 29 3.4 Cytotoxicity analysis of gelatin-PEI nanoparticles in HS68 cell line 29 3.5 His-eGFP delivery by gelatin-PEI nanoparticles in HS68 cell line 30 Chapter four Discussion 32 Chapter five Conclusion 35 Reference 5