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22대 대한민국 국회 국회정보길라잡이 국회의원검색
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Title Page 2

Abstract 5

Contents 7

Abbreviations 10

Ⅰ. Introduction 12

1. Direct Cell Reprogramming 12

2. Organoids and Assembloids 13

3. Drug-induced Liver Injury and Hepatic Fibrosis 14

4. Liver Organoids for Drug Screening and Toxicity Assessment 15

Ⅱ. Materials and Methods 16

1. Cell culture 16

2. Generation of liver assembloids 17

3. RNA extraction and cDNA synthesis 18

4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 18

5. Quantitative albumin secretion analysis 18

6. Immunofluorescence staining 18

7. Connective tissue staining 19

8. Cell viability assay 19

Ⅲ. Results 23

1. The Confirmation of Liver Markers of Liver Assembloid Cells 23

2. Generation of Liver Assembloids using Multi-lineage Cells 25

3. Characterization of Liver assembloids 28

4. Gene Expression Comparative Analysis of 2D Culture and Assembloids 29

5. Drug Toxicity Assessment in Liver Assembloids 31

Ⅳ. Discussion 35

Supplement Figure 38

References 39

List of Tables 9

Table 1. The primer list of qRT-PCR 20

Table 2. The antibody list of immunofluorescence staining 22

List of Figures 8

Figure 1. Principles of direct reprogramming and iPSCs technology 13

Figure 2. Overarching definition of an organoid along with the three sub-classifications 14

Figure 3. Application of liver organoids 16

Figure 4. Identifying of hepatic stem cells features of iHepSCs and HUVECs 24

Figure 5. Confirmation of cell features for assembloids generation 25

Figure 6. Generation of liver assembloids using multi-lineage cells 27

Figure 7. Confirmation of assembloid umformity 28

Figure 8. Characterization of liver assembloids 29

Figure 9. Comparative analysis of Liver cell marker genes expression in 2D culture and 3D... 30

Figure 10. Generation of hepatic fibrosis model using liver assembloids 32

Figure 11. Genetic analysis of fibrosis induced liver assembloids 33

Figure 12. Histologic analysis of fibrosis induced liver assembloids 34

List of Supplement Figures 8

Supplement Fig. 1. Fluorescence image of HUVEC-mKatke2 in assembloids 38

초록보기

 The liver is a major metabolic organ for drugs and exogeneous toxicants and can be damaged by a variety of stimuli, including alcohol and overdose of drugs. Drug-induced liver injury (DILI) is an acute or chronic liver disease caused by overexposure to drugs or compounds and is considered a critical factor in drug development and safety evaluation. This injury can lead to liver fibrosis and cirrhosis, which can progress to liver cancer. Therefore, hepatotoxicity assessment is essential to prevent liver injury. However, currently used DILI assessment tools have limitations, including a lack of cell diversity and technical difficulties in large-scale production. Consequently, developing an advanced platform for accurately predicting hepatic injury and fibrosis in vitro is imperative. In this study, we generated advanced liver assembloids that can be produced rapidly and in a large-scale manner using direct reprogrammed stem cells and cell lines. Furthermore, we confirmed the increase of collagen and cytochrome P450 (CYP) family, which are fibrosis markers, and proposed a tool to evaluate the hepatotoxicity of drug candidates.

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