Anti-apoptotic effect of 30Kc6 gene on induced pluripotent stem cell, and efficient delivery of 30Kc19-based transcription factor proteins for cellular direct conversion

다국어 초록 (Multilingual Abstract)

30K protein, derived from silkworm hemolymph (SH), was known as having many properties. From 30K family (30Kc6, 30Kc12, 30Kc19, 30Kc21, and 30Kc23), 30Kc6 plays role for anti-apoptosis. When the cells were transfected with 30Kc6, the cells had higher viability in stress condition. Also, these property of 30Kc6 could increase the productivity of antibody, recombinant interferon-β, and human erythropoietin (EPO). The mechanism of 30Kc6 for antiapoptosis was due to prevent Bax binding on mitochondria. The other 30K protein, 30Kc19, has reported the properties of cell-penetrating and enzyme-stabilizing. Cell-penetrating peptide (Pep-c19) of 30Kc19 could enter cells by forming dimer. Also, 30Kc19 stabilized enzyme by crowd effect. Recent, 30Kc19 protein was reported enhancing soluble expression for transcription factors. The objective of this research is to overcome several hurdles in stem cell research. Human pluripotent stem cells such as hES and hiPS cells suffer from apoptosis in single cell-dissociation. Because of pre-activated Bax at Golgi in cells, hES and hiPS cells induced rapid apoptosis in stress condition. We applied 30Kc6 in hiPS cells. hiPS-30Kc6 cells were expressed pluripotent stem cell markers, and had a potency of differentiation in three germ layers. Also, the viability of single cells was higher than normal hiPS cells. Then, we wondered that anti-apoptotic property of 30Kc6 was from a specific domain of 30Kc6. From the apoptosis research, BH4 domain of Bcl2 inhibited apoptosis by binding Bax. With structural similarity of BH4, 30Kc6 alpha-helix (30Kc6α) was considered as anti-apoptotic domain. By truncation, we cloned 30Kc6α and observed anti-apoptotic property. Furthermore, 30Kc19-30Kc6α protein could penetrate cell, and showed enhanced cellular viability under STS or UV-irradiation condition. Cell-penetration and enzyme-stabilization are the properties on 30Kc19 protein. We tried to solve the problems on reprogramming which are instability and low solubility of transcription factor proteins. Previous reprogramming methods had a risk of transgene integration to host genome. By using protein, transcription factors were delivered directly without any DNA-related complications. To express transcription factors as soluble form, 30Kc19 was conjugated. 30Kc19 conjugated transcription factors (Oct4, Sox2, c-Myc, and Klf4) were expressed as a soluble protein. Purified soluble transcription factors were penetrated into cells, and stable up to 48 h. Furthermore, Klf4-30Kc19 protein showed a transcriptional activity. Lastly, we generated neuronal cells using transcription factor with 30Kc19. 30Kc19-Ascl1 protein induced successfully mouse embryonic fibroblasts (MEFs) to protein-induced neuronal cells (piNCs). p-iNCs had neuronal morphology, and expressed neuronal markers (Tuj1 and MAP2). Furthermore, the expression levels of neuronal genes (ASCL1, BRN2, and MYT1L) were observed higher on day 7 and 14. In this research, we applied anti-apoptotic 30Kc6 and cellpenetrating 30Kc19 protein on stem cell research. This approach will give a chance for developing new techniques in stem cell research.

목차 (Table of Contents)

• Chapter 1. Research background and objective ................................. 1
• Chapter 2. Literature review ............................................................... 6
• 2.1 Cellular reprogramming .......................................................... 7
• 2.2 Reprogramming methods ........................................................ 9
• 2.2.1 Virus .................................................................................. 9
• 2.2.2 DNA ..................................................................................10
• 2.2.3 mRNA and miRNA .......................................................... 13
• 2.2.4 Cre/LoxP and PiggyBac transposon .............................. 14
• 2.2.5 Protein ............................................................................. 16
• Chapter 3. Experimental procedures ................................................ 18
• 3.1 Gene cloning .......................................................................... 19
• 3.2 Protein production and analysis ........................................... 19
• 3.2.1 Protein expression and purification …............................ 19
• 3.2.2 Quantitative analysis using BSA standard solution ...... 20
• 3.2.3 Western blot analysis for in vitro stability assay ......... 21
• 3.3 Cell culture ..........................................................................… 22
• 3.3.1 Primary cell isolation and culture .................................. 22
• 3.3.2 Embryonic stem cell culture and embryoid body
• formation ............................................................................ 23
• 3.4 Generation of induced pluripotent stem cell (iPSC) ......…… 24
• 3.4.1 Retrovirus production ..................................................... 24
• 3.4.2 Viral transduction and iPSC culture ............................... 24
• 3.5 Generation of protein-induced neuronal cell (p-iNC) ....... 27
• 3.6 Cell analysis ........................................................................... 27
• 3.6.1 RNA isolation and cDNA synthesis ............................... 27
• 3.6.2 RT-PCR and RT-qPCR ................................................. 28
• 3.6.3 Immunocytochemistry .................................................... 31
• 3.6.4 Live cell analysis ............................................................ 31
• 3.6.5 Cell viability assay .......................................................... 32
• 3.6.6 Luciferase assay ............................................................ 32
• 3.6.7 Apoptosis induction and FACS analysis ........................ 33
• 3.6.8 Single cell-dissociation and Alkaline phosphatase
• staining .............................................................................. 34
• 3.6.9 Electrophysiological recording ………............................... 35
• Chapter 4. Anti-apoptotic effect of 30Kc6 gene on human induced
• pluripotent stem cell ........................................................................... 36
• 4.1 Introduction ........................................................................... 37
• 4.2 Retroviral transduction and expression of hESC-specific
• genes in hiPSC-30Kc6 ........................................................ 40
• 4.3 In vitro differentiation of hiPSC-30Kc6 ................................. 44
• 4.4 Anti-apoptosis effect on hiPS-30Kc6 cells ....................... 46
• 4.5 Single cell-dissociated cell viability .................................... 49
• 4.6 Conclusions ............................................................................. 52
• Chapter 5. Anti-apoptotic role of 30Kc6 alpha helix domain and its
• application by conjugating with 30Kc19 protein ............................... 53
• 5.1 Introduction ............................................................................ 54
• 5.2 Cloning of 30Kc6α and gene expression in
• mammalian cells ...................................................................... 57
• 5.3 Anti-apoptosis property of 30Kc6α .................................. 59
• 5.4 Expression and purification of 30Kc19-30Kc6α protein
• ............................................................................................ 62
• 5.5 Cytotoxicity and cell penetration of 30Kc19-30Kc6α
• protein ................................................................................... 62
• 5.6 Anti-apoptosis property of 30Kc19-30Kc6α protein ..... 63
• 5.7 Conclusions ………………………..……………………….……………………….…. 67
• Chapter 6. Soluble expression and stability enhancement of
• transcription factors using 30Kc19 cell-penetrating
• protein .................................................................................................. 69
• 6.1 Introduction ………………………………………………………….….……………… 70
• 6.2 Soluble expression and purification of transcription
• factors ………………………………………………………………………….…………... 74
• 6.3 Analysis of purified soluble transcription factors ............... 78
• 6.4 In vitro stability of soluble transcription factors ................ 78
• 6.5 Cell penetration and intracellular stability of soluble
• transcription factors ………………………..................................... 82
• 6.6 Cytotoxicity of 30Kc19-conjugated transcription factors
• ........................................................................................... 83
• 6.7 Transcriptional activity of 30Kc19-conjugated Klf4 ........ 86
• 6.8 Conclusions ........................................................................... 87
• Chapter 7. Direct conversion of fibroblasts to neuronal cells by
• 30Kc19-Ascl1-NLS-R9 protein ..................................................... 91
• 7.1 Introduction ………………………………………………………….…….………….. 92
• 7.2 Soluble expression of 30Kc19-Ascl1-NLS-R9 protein
• ........................................................................................... 93
• 7.3 Purification, and in vitro stability of protein …………….……….. 96
• 7.4 Cell penetration ……………………………………………………….…...…..….. 98
• 7.5 Cytotoxicity assay ………………………………………………………….…… 100
• 7.6 Generation of protein-induced neuronal cells (p-iNCs)
• ......................................................................................... 100
• 7.7 Cell morphology change of p-iNCs …………………………...……. 103
• 7.8 Expression of neuronal biomerkers in p-iNCs ……….……… 103
• 7.9 Neuronal gene expression in p-iNCs …………………….…..…... 106
• 7.10 Electrophysiological recordings …………………….…..………..... 106
• 7.11 Conclusions ………………………………………………………..………...…… 110
• Chapter 8. Overall discussion and further suggestions ................. 112
• Bibliography ...................................................................................... 119
• Korean abstract ................................................................................ 132