Confirmation of human mesenchymal stem cell cultivation without differentiation

국문 초록 (Abstract)

중간엽 줄기세포는 성인줄기세포로서 뼈, 연골, 지방 같은 중간엽 조직의 재생에 관여한다. 최근의 연구에 따르면 이 세포들은 다른 기원에 있는 세포로 분화되기도 하는데 이것은 세포치료에서 다양한 질병들을 치료하는데 방대한 원천을 제공한다. 그러나 이 세포들은 골수에서 매우 적은 수가 있고 그래서 치료하기 전 in vitro에서 많은 증식을 필요로 한다. 이 논문에서 우리는 자발적인 분화 없이 미분화 상태로 몇 개대까지 증식이 가능한지 또 2-DE를 이용해 각 개대별 미분화 중간엽 줄기세포의 단백질 차이를 보여주고자 하였다. 결론적으로 4, 7, 8 개대에서 뼈모세포로 자발적인 분화가 일어나지만 그 비율은 매우 적고 그래서 8개대까지 미분화상태의 중간엽 줄기세포를 증식 할 수 있었다.

다국어 초록 (Multilingual Abstract)

Mesenchymal stem cells(MSCs) are adult stem cells, which contribute to the regeneration of the mesenchymal tissues such as bone, cartilage, and adipose. Recent studies suggest that these cells also have the potentials to differentiate into other lineage cells, thereby providing ample sources for treating the various disease in the cell therapy modality. However, these cells are such small population in the aspirated bone marrow that necessitates in vitro expansion step prior to the treatment. In this study, therefore, I aim to show how many passages are possible to c ltivate human MSCs in the undifferentiated state without the spontaneous differentiation. Results showed that hMSCs from the passage 4,7 and 8 spontaneously differentiated to the osteoblast as confirmed by the expression of alkaline phosphatase. But portion of differentiated hMSCs was very small and we could proliferate hMSCs until passage 8 because of slowing growth rate. Different spots of hMSCs proteome at each passage compared to passage 2 existed, but we couldn’t detect meaningful spots. We postulated that this result was induced by experimental vatiation. We anticipated that hMSCs were used in many field without suspect of differentiation and potentiality loss until passage 8.

목차 (Table of Contents)

• Table of Contents
• List of Tables = ⅰ
• List of Figures = ⅱ
• Abbreviations = ⅲ
• Abstract = ⅳ
• Ⅰ. Introduction = 1
• Ⅱ. LITERATURE REVIEW = 2
• 2-1. What are MSCs? = 2
• 2-2. Mesoderm = 3
• 2-3. CD90 (Thy-1) = 3
• 2-4. CD44 = 3
• 2-5. CD34 = 4
• 2-6. Osteopontin = 5
• 2-7. LPL(Lipoprotein lipase) = 6
• 2-8. Alkaline phosphatase = 6
• 2-9. Collagen type Ⅱ = 7
• 2-10. GAPDH = 7
• Ⅲ. MATERIALS AND METHODS = 9
• 3-1. Isolation and culture of undifferentiated hMSCs = 9
• 3-2. Flow cytometry = 10
• 3-3. Reverse transcription (RT) and PCR = 10
• 3-4. Inducing differentiation from hMSCs to osteocyte and adipocyte = 10
• 3-5. Alizarin Red S staining = 11
• 3-6. Oil Red O staining = 11
• 3-7. Tow dimensional gel electrophoresis =12
• 3.7.1. Sample preparation = 12
• 3.7.2. Isoelectic focusing (IEF) = 12
• 3.7.3. Equilibration of IEF gel strips = 13
• 3.7.4. SDS-PAGE = 13
• 3.7.5. Silver Staining = 13
• 3.7.6. Image Analysis = 14
• 3.8. MALDI-TOF analysis for proteins = 14
• 3.8.1. Sample preparation for MALDI-TOF = 14
• 3.8.2. MALDI-TOF mass spectrometry = 15
• Ⅳ. Result = 17
• 4.1. Growth rate of hMSCs from passage 2 to passage 8 = 17
• 4-2. Characterization of hMSCs by RT-PCR from passage 2 to passage8 = 19
• 4.3. Confirmation of characterization and potentials of defferentiation of hMSCs by inducing media using Alizarin Red S, Oil Red O and RT-PCR = 21
• 4.4. Characterization of hMSCs by FACS at passage 3 and 8 = 26
• 4.5. Proteome difference of hMSCs from passage 2 to passage 7 = 29
• Ⅴ. Discussion = 34
• Ⅵ. Reference. = 37
• Ⅶ. Abstract in Korean = 42