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間質幹細胞於臨床再生醫學應用之研究
Other Title
Potential Clinical Application of Mesenchymal Stem Cells for Regenerative Medicine
Type
thesis
Date Issued
2016-07-21
Author(s)
林宏達
Advisor
歐耿良
Subjects
系所名稱：生醫材料暨組織工程研究所
Description
學位別：碩士
語文別：英文
指導教授：歐耿良
共同指導教授：
口試委員：劉沖明;陳錦松
中文關鍵字：牙齦組織, 富血小板血漿幹細胞;骨再生
英文關鍵字：gingival tissue, platelet-rich plasma stem cells;bone regeneration
語文別：英文
指導教授：歐耿良
共同指導教授：
口試委員：劉沖明;陳錦松
中文關鍵字：牙齦組織, 富血小板血漿幹細胞;骨再生
英文關鍵字：gingival tissue, platelet-rich plasma stem cells;bone regeneration
Abstract
Background
Recent advances in stem cell research have prompted the development of cell-based therapies for a series of common diseases, including osteoporosis, osteoarthritis, Parkinson’s disease, diabetes and Alzheimer’s disease. However, the isolation of BMSCs may be painful to the donor, requiring spinal anesthesia and may yield low numbers of MSCs. To overcome these problems, we aim to identify and utilize an alternative stem cells derived from gingival tissue termed as gingival stem cells (GSCs).
Although certain researches had shown that platelet-rich plasma (PRP) could promote the osteogenic differentiation and might have the potential on bone regeneration, the applications of PRP on induction of GSCs are still rare and the effect of osteogenic differentiation is still unknown.
Aims
The aim of this study is to evaluate the effect of PRP and GSCs on in vitro osteogenic differentiation.
Material and Methods
GSCs were harvest from gingival tissue and incubated overnight at 4°C with dispase (2 mg/mL). After that we separated the epithelial and lower spinous layers which were then digested in collagenase IV (4 mg/mL) for 4 hours. The single cell suspension was filtered through a 70-μm cell strainer. These cell suspensions were plated and cultured in alpha-MEM with 10% fetal bovine serum and treated with 5 % PRP. After 7 days, the cells were harvested and analyzed by qPCR to determine osteogenic differentiation genes. These cells were certified and examined for their stem cells markers such as CD34, CD45, CD73, CD90 and CD105. Cell growth (doubling time and colony formation) were studied to analyze the isolated stem cells at passage number 3, for measuring the function played by PRP on GMSCs capacity to undergo osteogenic differentiation. qPCR and immunofluorescence staining (OCN and F-actin) were done for observing the results.
Results
Flow cytometry was conducted to identify expression of stem cell specific markers in GSCs. Isolated stem cells demonstrated positive expression of positive (CD73, CD90 and 105) and negative stem cell markers (CD34 and CD45). Thus, we could make sure that our stem cells are of the correct type and accounted for a high proportion, and from the results we demonstrated that isolated cells have good cell potential of cell proliferation (doubling time:3.41 hrs) and colony formation. In addition, we also found that PRP treatment is noncytotoxic to GSCs at 5% cultured, and osteogenic gene expression (ALP and OCN) is significantly up-regulated. Immunofluorescence staining also indicated that significantly higher positive expression of cytoskeleton and osteogenic markers, F-Actin and OCN after PRP treatment.
Conclusions
GSCs can be isolated and expanded when applying appropriate procedures. They preserved their capacity to differentiate by using PRP and therefore provide a convenient alternative source of stem cells for regenerative medicine
Recent advances in stem cell research have prompted the development of cell-based therapies for a series of common diseases, including osteoporosis, osteoarthritis, Parkinson’s disease, diabetes and Alzheimer’s disease. However, the isolation of BMSCs may be painful to the donor, requiring spinal anesthesia and may yield low numbers of MSCs. To overcome these problems, we aim to identify and utilize an alternative stem cells derived from gingival tissue termed as gingival stem cells (GSCs).
Although certain researches had shown that platelet-rich plasma (PRP) could promote the osteogenic differentiation and might have the potential on bone regeneration, the applications of PRP on induction of GSCs are still rare and the effect of osteogenic differentiation is still unknown.
Aims
The aim of this study is to evaluate the effect of PRP and GSCs on in vitro osteogenic differentiation.
Material and Methods
GSCs were harvest from gingival tissue and incubated overnight at 4°C with dispase (2 mg/mL). After that we separated the epithelial and lower spinous layers which were then digested in collagenase IV (4 mg/mL) for 4 hours. The single cell suspension was filtered through a 70-μm cell strainer. These cell suspensions were plated and cultured in alpha-MEM with 10% fetal bovine serum and treated with 5 % PRP. After 7 days, the cells were harvested and analyzed by qPCR to determine osteogenic differentiation genes. These cells were certified and examined for their stem cells markers such as CD34, CD45, CD73, CD90 and CD105. Cell growth (doubling time and colony formation) were studied to analyze the isolated stem cells at passage number 3, for measuring the function played by PRP on GMSCs capacity to undergo osteogenic differentiation. qPCR and immunofluorescence staining (OCN and F-actin) were done for observing the results.
Results
Flow cytometry was conducted to identify expression of stem cell specific markers in GSCs. Isolated stem cells demonstrated positive expression of positive (CD73, CD90 and 105) and negative stem cell markers (CD34 and CD45). Thus, we could make sure that our stem cells are of the correct type and accounted for a high proportion, and from the results we demonstrated that isolated cells have good cell potential of cell proliferation (doubling time:3.41 hrs) and colony formation. In addition, we also found that PRP treatment is noncytotoxic to GSCs at 5% cultured, and osteogenic gene expression (ALP and OCN) is significantly up-regulated. Immunofluorescence staining also indicated that significantly higher positive expression of cytoskeleton and osteogenic markers, F-Actin and OCN after PRP treatment.
Conclusions
GSCs can be isolated and expanded when applying appropriate procedures. They preserved their capacity to differentiate by using PRP and therefore provide a convenient alternative source of stem cells for regenerative medicine