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Potential of secretome of fetal cartilage progenitor cells on regeneration of tissue; application to wound healing, hair growth and osteoarthritis

초록/요약

Although tissue repair process is well known as characterization by four overlapping phases, including an early reaction to preserve homeostasis, an inflammatory reaction to protect against infection, a proliferative phase to rebuild the wound site, and a remodeling phase to restore tissue strength and function when tissue repair process is completed. In addition, all cells in each tissue of organs communicate with each other and are linked to the tissue repair process. Therefore, a drug that can act on all these tissues would be ideal. Theoretically, no single molecule can control inflammation and metabolism by acting on all cells in this manner. To date, many methods have been attempted to prevent the progression of damaged tissue, including pharmacological and nonpharmacological treatments, but none have been proven to be clinically effective yet. Therefore, biological therapies using cells and/or their secretome are expected to have considerable potential in this respect. Recent studies have shown that the use of MSC secretome has been assessed to enhance the clinical applicability of this biological treatment as a true tissue repair agent/drug and to overcome the disadvantages of cell-based therapy while maintaining its advantages to their parent cells. Human fetal cartilage progenitor cells-secretome (ShFCPC) has shown potent anti-inflammatory and tissue-repair effects better than that of mesenchymal stem cells-secretome. However, its underlying mechanisms and effects on tissue repair process have rarely been systematically elucidated. Thus, the purpose of this study was to isolate and develop a ready-to-use product, novel non-immunogenic ShFCPC for clinical application, analyze molecular components within ShFCPC, and verify its tissue repair therapeutic efficacy. In chapter I, we present a fabrication technology, including 3D pellet culture, filtration and lyophilization to produce a ready-to-use product, novel non-immunogenic ShFCPC containing superior quantity and quality of ECM molecules and bioactive molecules involving many cellular processes essential to homeostasis including the cell cycle, cell survival, inflammation, metabolism, and apoptosis. In addition, we first analyzed the secretome released by hFCPC, which was established by constructing, for the first time, an extensive map of the ShFCPC for supplying information on how they interact with biological pathways in tissue repair process. Thus, we suggest that ShFCPC could be a potential anti-inflammatory and anti-apoptotic agent for various stress microenvironment-related disorders. In chapter II, we determined the potential utility of the identified proteins involved in inflammatory diseases such as osteoarthritis (OA) protection and validated their biological functions both in vitro and in vivo in an OA model are compared with those of human bone marrow-derived mesenchymal stem cells-secretome (ShBMSC) and hyaluronan (HA). Biological validation in vitro has shown that ShFCPC protects chondrocyte apoptosis by suppressing the expression of inflammatory mediators and matrix-degrading proteases and promotes the secretion of pro-chondrogenic cytokines in lipopolysaccharide-induced coculture of human chondrocytes and SW982 synovial cells compared with ShBMSC. Moreover, in a rat OA model, ShFCPC protects articular cartilage by reducing inflammatory cell infiltration and M1/M2 macrophage ratio in the synovium, by reducing inflammatory cell infiltration and M1/M2 macrophage ratio in the synovium, cartilage repair compared to ShBMSC and HA. Our findings support clinical translations of ShFCPC as a novel agent for modifying OA process. In chapter III, as an extended clinical application of ShFCPC, we hypothesized that anti-apoptotic and tissue repair effects of ShFCPC could be suitable properties for hair loss treatment. Its useful properties are compared with those of minoxidil in terms of in vitro studies of dermal papilla (DP) cells behavior, hair follicle germ (HFG) formation, and ex vivo hair transplantation and in vivo hair loss model. hFCPC can alleviate hair loss by attenuating catabolism and apoptosis of DP cells. Furthermore, ShFCPC treatment is an effective therapeutic strategy to improve the hair repair process by cell-cell interactions between DP cells and epidermal cells creating a favorable environment for DP cells; this alters the local microenvironment and stimulates the activation of 𝛽-catenin signaling of DP cells repairing the damaged hair follies. These findings suggest that ShFCPC may be a novel regenerative agent suitable for hair repair and hair loss treatment.

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목차

BACKGROUND 1
1.1. Secretome as a regenerative therapeutic agent 2
1.2. Bio-inductive components of secretome 2
1.3. Preparation of stem cell secretome 3
1.3.1. Approaches to produce stem cell secretome 3
1.3.2. Characterization of stem cell secretome 4
1.3.3. Novel strategies to increase therapeutic efficacy and production of stem cell secretomes 4
1.4. Clinical applications of secretome-based biomedicines 5
1.5. Considerations for secretome-based biomedicines research 6
1.6. Fetal stem cell-derived secretome 7
1.7. Thesis overview 9
CHAPTER I: Preparation and analysis of secretome of human fetal cartilage progenitor cells for tissue repair application 11
2.1. Introduction 12
2.2. Materials and methods 14
2.2.1. Preparation of ShFCPC 14
2.2.2. SDS‑PAGE and In‑gel digestion 15
2.2.3. LC–MS/MS analysis 15
2.2.4. MS data analysis 16
2.2.5. Reverse transcription-polymerase chain reaction (RT-PCR) 17
2.2.6. Enzyme-linked immunosorbent assay (ELISA) 18
2.2.7. Western blot assay 18
2.2.8. Statistical analysis 19
2.3. Results 20
2.3.1. Preparation of ShFCPC 20
2.3.2. LC–MS/MS based analysis of diferentially expressed proteins in ShFCPC and ShBMSC 21
2.3.3. Secretome interaction network of functional enrichment pathway in ShFCPC and ShBMSC 22
2.3.4. Validation of LC MS/MS identified proteins of the ShFCPC and ShBMSC 24
2.3.5. Secreted proteins enriched in ShFCPC regarding cellular processes 26
2.4. Discussion 32
CHAPTER II: Potential of secretome of human fetal cartilage progenitor cells as disease modifying agent for osteoarthritis 35
3.1. Introduction 36
3.2. Materials and methods 39
3.2.1. Evaluation of chondrocytes viability, proliferation, and migration 39
3.2.2. Coculture of inflamed chondrocytes and synovial cell as in vitro model for osteoarthritis 40
3.2.3. Animals and OA induction 41
3.2.4. Experimental groups and treatment 42
3.2.5. Reverse transcription-polymerase chain reaction (RT-PCR) 43
3.2.6. Enzyme-linked immunosorbent assay (ELISA) 44
3.2.7. Histopathological analysis 45
3.2.8. Immunohistochemistry 48
3.2.9. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining 48
3.2.10. Statistical analysis 49
3.3. Results 50
3.3.1. Dose-dependent effect of ShFCPC on chondrocyte viability, proliferation, and migration 50
3.3.2. Effects of ShFCPC on inflamed chondrocyte-SW982 in a coculture system 53
3.3.3. Osteoarthritis induction in rat model using combined intra-articular injection of collagenase with rigorous exercise involving a specific amount of running 55
3.3.4. ShFCPC promoting repair of injured articular cartilage in OA rats 58
3.3.5. Effects of ShFCPC on the inflammatory cellular changes of synovium in OA rat 60
3.3.6. ShFCPC alleviation of the progression of OA in vivo by reducing the levels of pro-inflammatory cytokines and ECM proteins degradation in body fluids 63
3.4. Discussion 66
CHAPTER III: Secretome of human fetal cartilage progenitor cells as a regenerative agent for testosterone-induced hair loss 70
4.1. Introduction 71
4.2. Materials and methods 74
4.2.1. Animals 74
4.2.2. Preparation of human DP cells and murine epithelial cells 74
4.2.3. Effects of a ShFCPC dose on DP cells viability, proliferation, and migration 75
4.2.4. Effects of minoxidil and testosterone on human DP cells number 75
4.2.5. Effects of ShFCPC on testosterone-induced DP cells apoptosis in vitro 76
4.2.6. Spontaneous hair follicle germ (HFG) formation 77
4.2.7. Effects of ShFCPC on HFG formation under testosterone stimulation 77
4.2.8. Ex vivo hair regeneration assay 78
4.2.9. Application to hair growth in vivo 80
4.2.10. Determination of hair morphology 81
4.2.11. Histomorphometric analysis in vivo 81
4.2.12. Reverse transcription-polymerase chain reaction (RT-PCR) 82
4.2.13. Flow cytometry analysis 83
4.2.14. Western blot assay 84
4.2.15. Immunocytochemistry 84
4.2.16. Immunohistochemistry 85
4.2.17. Statistical analysis 87
4.3. Results 88
4.3.1. Dose-dependent effect of ShFCPC on DP cell viability, proliferation, and migration 88
4.3.2. Effects of minoxidil and testosterone on human DP cells number 91
4.3.3. Effects of ShFCPC on the biological properties of testosterone-induced DP cells 93
4.3.4. Validation of freshly isolated epithelial cells 95
4.3.5. Effects of different cell densities on HFG formation and trichogenic gene expression and HF regeneration efficiency 97
4.3.6. Effects of ShFCPC on HFG formation under testosterone stimulation 102
4.3.7. ShFCPC promotes ex vivo hair follicle generation under testosterone stimulation 104
4.3.8. Hair regeneration effects of ShFCPC in a testosterone‑induced hair loss rat model 108
4.3.9. ShFCPC treatment alters expression of signaling molecules in hair follicles in vivo 110
4.4. Discussion 112
CONCLUSIONS 117
REFERENCE 119

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