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Study on control of stem cell differentiation and tissue regeneration by synthetic peptide : 합성 펩타이드에 의한 줄기세포 분화 조절 및 조직재생 연구

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Authors

Dong Woo Lee

Advisor
박윤정
Major
치의학대학원 치의과학과
Issue Date
2017-02
Publisher
서울대학교 대학원
Keywords
stem cellsynthetic peptidereprogrammingiPSCBone regenerationCPNE7
Description
학위논문 (석사)-- 서울대학교 대학원 : 치의과학과, 2017. 2. 박윤정.
Abstract
Stem cells are undifferentiated cells that can infinitely regenerate and differentiate into cells of all tissues in the body. Currently, research on tissue regeneration using adult stem cells is being actively conducted in the field of regenerative medicine. However, due to the limitations of extracorporeal proliferation, it is difficult to obtain a sufficient amount of adult stem cells. In addition, their differentiation ability is relatively low, which is a further disadvantage. To solve this problem, induced pluripotent stem cells (iPSCs) with the characteristics of embryonic stem cells have been developed by the Yamanaka group by dedifferentiating and reprogramming differentiated somatic cells. Because iPSCs are produced from somatic cells of the adult, there are no ethical issues, and their ability to self-reproduce is excellent, allowing a large number of cells to be supplied. However, the currently representative iPSCs manufacturing techniques cannot risk using viruses to alter cells, and thus, the cells must be altered by proteins or other methods . However, proteins have been demonstrated to exhibit low cell reprogramming efficiency. To improve the reprogramming efficiency, essential reaction conditions for reverse reprogramming have been explored. In this study, experiments were conducted to promote reprogramming using synthetic peptides.
To apply iPS cells and stem cells in the tissue regeneration field, differentiation into the final tissues, such as bone and cartilage, must be performed.
Recently, it has been reported that NF-κB promotes epithelial-mesenchymal transition (EMT), and mesenchymal-epithelial transition (MET) is essential for reprogramming cells to form iPSCs. To improve the reprogramming efficiency, which is a useful cell therapy technique for clinical application of iPSCs, functional peptides inhibiting NF-κB activity have been identified. It was confirmed that these peptides activated MET by inhibiting NF-kB, eventually promoting cellular reprogramming. The Yamanaka factors were injected into human dermal fibroblasts using a nucleofection method, and then, cells were further treated with the synthetic peptides.
In the peptide-treated groups, the expression levels of MET markers and pluripotency markers were higher than in the negative control group, and it was confirmed that relatively more iPS cells were formed at an early stage in the peptide-treated groups. At the same time, NF-κB expression was decreased. Thus, it might be considered that functional peptides have been shown to inhibit the translocation of intracellular NF-κB into the nucleus, thereby inhibiting EMT, resulting in the promotion of the opposite process, MET, followed by enhancement of reprogramming efficiency.

Recently, tissue regeneration through stem cell differentiation has been attempted in the dental field. It has been verified through various experiments that the protein copine 7 (CPNE7) is a key factor for differentiating dental pulp stem cells (DPSCs) into dentin. It is also known that non-dentin mesenchymal stem cells can be differentiated into dentin and cementum using CPNE7 protein. CPNE7 has been applied for dentin regeneration. However, in this study, CPNE7 was employed as a bone forming substance. In addition, the identification of key amino acid sequences of CPNE7 was of interest in this study. In this study, a CPNE7-derived peptide was used to maximize biocompatibility and increase the regeneration of the jawbone and gum bones in the elderly.
CPNE7 consists of two domains, a C2 domain and a von Willebrand factor type A (vWA) domain. The C2 domain targets the extracellular lipid membrane and promotes intracellular transfer of specific proteins. It is also known as a domain that is stabilized through the binding of 2 ~ 3 calcium ions. The vWA domain is known to mediate protein-protein interactions. Based on these domains characteristics, 6 functional peptides sequences were designed and tested to determine if any portion of the peptide could replace the activity of CPNE7.
The cytotoxicity and translocation ability of the candidate peptides derived from CPNE7 were examined. The expression of osteogenic protein factors was also measured. The peptides derived from CPNE7 were named CPNE7-derived peptides (CDPs). Among the candidate peptides, peptides that enabled an increase in osteogenic protein markers were selected based on mineral staining assays with alizarin red S and alkaline phosphatase assays. Furthermore, in an animal model, collagen, a representative biocompatible scaffold, was mixed with a CDP or a comparative group protein, and then, the mixtures were transplanted into a calvarial defect mouse model to confirm bone regeneration.
In the future, application of such peptide-based stem cell differentiation inducers and associated reprogramming control techniques will be helpful for tissue regeneration therapy and cell therapy.
Language
English
URI
https://hdl.handle.net/10371/130925
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