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Stem cell properties of human fetal cartilage derived progenitor cells as a novel cell source of regenerative medicine

Stem cell properties of human fetal cartilage derived progenitor cells as a novel cell source of regenerative medicine

초록/요약

Stem cells research in tissue engineering and regenerative medicine is likely to offer a second generation of therapy for clinical diseases that cannot be treated until now. The stem cells possess two properties, self-renewal and differentiation potential. Differentiation potential of stem cells is the ability to differentiate into specialized cells, in response to appropriate signals. Because of ability to differentiate into different types of functional cells. MSCs are especially the major stem cells for regenerative medicine about approximately 10 years. Because MSCs can be obtained from a variety of tissues.But during long term expansion in vitro, they have disadvantages in terms of decrease differentiation and self-renewal ability as well as characteristic changes of senescence. To application of stem cells in regenerative medicine, in vitro expansion is required to reach the large cell numbers and maintain their character are necessary in order to use the cells. To generate therapeutically safe and usable stem cell-derived products for clinical cell therapies, all animal-derived material must be eliminated from the establishment, culture, and differentiation steps. In the stem cell therapies, the animal-derived reagents was a limitation and risk so in the most researches xeno free conditions is the best choice that do not contain animal components. Elimination of animal components during the in vitro long-term expansion as well as differentiation of stem cells is necessary prior to application in cell therapy. Previously, we demonstrated that characteristics of human fetal cartilage-derived cells (H.FCPCs) as a stem cells in comparison with H.MSCs and young chondrocytes in terms of their proliferation ability and plasticity in vitro and in vivo . And we confirmed that it is a cell source with excellent colony forming ability and no tumorigenicity in vivo.(not published) As well as, H.FCPCs showed low toxicity and immune rejection when injected in a rat model, and the possibility of treatment of synovial arthritis was confirmed.

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목차

Abstract----------------------------------------------------------------------------------------------I
Contents--------------------------------------------------------------------------------------------IV
List of Figures------------------------------------------------------------------------------------ VII

Background ------------------------------------------------------------------------------------------1
1. Stem Cells and Regenerative Medicine -----------------------------------------------------1
1.1 Types of stem cells used in regenerative medicine -------------------------------------2
1.1.1 Embryonic stem cells (ESCs)--------------------------------------------------------------2
1.1.2 Mesenchymal stem cells (MSCs) --------------------------------------------------------2
1.1.3 Induced pluripotent stem cells (iPSCs)-------------------------------------------------3
1.1.4 Fetal cells (FCs) -----------------------------------------------------------------------------3
2. What is the Fetal cartilage derived stem cells----------------------------------------------5
3. Stem cells and Self-renewal ------------------------------------------------------------------6
3.1 Self –renewal ----------------------------------------------------------------------------------6
4. Senescence --------------------------------------------------------------------------------------7
4.1 Replicative Cellular Senescence ------------------------------------------------------------9
5. Culture conditions optimization of stem cells -------------------------------------------12
6. Aims of this study-----------------------------------------------------------------------------16

Chapter I. -----------------------------------------------------------------------------------------17
1. Introduction-----------------------------------------------------------------------------------17
2. Materials and Methods----------------------------------------------------------------------20
2.1 Isolation and culture of Human fetal cartilage derived progenitor cells----------20
2.2 Colony forming unit fibroblast (CFU-F) assay -----------------------------------------20
2.3 Cellular Senescence Assay-----------------------------------------------------------------21
2.4 RNA Isolation and Reverse Transcription-Polymerase Chain Reaction------------21
2.5 In vivo Tumorigenesity---------------------------------------------------------------------21
2.6 Histological Evaluation --------------------------------------------------------------------22
2.7 Differentiation potential-------------------------------------------------------------------22
2.8 Microarray -----------------------------------------------------------------------------------23
3. Results------------------------------------------------------------------------------------------24
3.1 Colony formation ability of H.FCPCs ---------------------------------------------------24
3.2 Cellular senescence of H.FCPCs long term culture ----------------------------------26
3.3 h.TERT Gene Expression of H.FCPCs ---------------------------------------------------27
3.4 Self –renewal related gene RT-PCR------------------------------------------------------29
3.5 Multilinage differentiation ability -------------------------------------------------------31
3.6 In vivo Tumorigenesity --------------------------------------------------------------------33
3.7 Microarray -----------------------------------------------------------------------------------35
4. Discussion--------------------------------------------------------------------------------------37

Chapter II. -----------------------------------------------------------------------------------------41
1. Introduction------------------------------------------------------------------------------------41
2. Materials and Methods----------------------------------------------------------------------44
2.1 Isolation and culture of Human fetal cartilage derived progenitor cells----------44
2.2 Cell proliferation ability---------------------------------------------------------------------44
2.3 Colony forming unit fibroblast (CFU-F) assay ------------------------------------------45
2.4 RNA Isolation and Reverse Transcription-Polymerase Chain Reaction ------------45
2.5 Flow cytometric Analysis-------------------------------------------------------------------45
2.6 Cellular Senescence Assay-----------------------------------------------------------------46
2.7 Differentiation potential-------------------------------------------------------------------46
2.8 Cytogenic analysis of H.FCPCs------------------------------------------------------------47
3. Results------------------------------------------------------------------------------------------48
3.1 Long-term Proliferation ability of H.FCPCs --------------------------------------------48
3.2 Cellular senescence of H.FCPCs ---------------------------------------------------------50
3.3 Colony formation ability of H.FCPCs----------------------------------------------------52
3.4 Expression of stem cell-related genes in H.FCPCs -----------------------------------54
3.5 Multilinage differentiation ability of H.FCPCs-----------------------------------------58
3.6 Chromosomal stability of H.FCPCs after long-term culture-------------------------61
4. Discussion--------------------------------------------------------------------------------------63
Conclusion ---------------------------------------------------------------------------------------67
References-----------------------------------------------------------------------------------------69
국문요약--------------------------------------------------------------------------------------------76

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