Transdifferentiation of Human Mesenchymal Stem Cells Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Tatjana Schilling aus San Miguel de Tucuman, Argentinien Würzburg, 2007 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Martin J. Müller Gutachter: PD Dr. Norbert Schütze Gutachter: Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig angefertigt und keine anderen als die von mir angegebenen Hilfsmittel und Quellen verwendet habe. Des Weiteren erkläre ich, dass diese Arbeit weder in gleicher noch in ähnlicher Form in einem Prüfungsverfahren vorgelegen hat und ich noch keinen Promotionsversuch unternommen habe. Gerbrunn, 4. Mai 2007 Tatjana Schilling Table of contents i Table of contents 1 Summary ........................................................................................................................ 1 1.1 Summary.................................................................................................................... 1 1.2 Zusammenfassung..................................................................................................... 2 2 Introduction.................................................................................................................... 4 2.1 Osteoporosis and the fatty degeneration of the bone marrow..................................... 4 2.2 Adipose and bone tissue ............................................................................................ 4 2.2.1 Adipose tissue......................................................................................................4 2.2.2 Bone tissue........................................................................................................... 5 2.2.3 Bone remodelling..................................................................................................7 2.3 Mesenchymal stem cells ............................................................................................ 8 2.3.1 Multilineage potential............................................................................................8 2.3.2 Adipogenesis........................................................................................................9 2.3.3 Osteogenesis ..................................................................................................... 11 2.3.4 Inverse relationship between adipogenic and osteogenic differentiation............. 14 2.4 Transdifferentiation................................................................................................... 15 2.5 Objective of this study .............................................................................................. 17 3 Materials and methods ................................................................................................ 18 3.1 Materials .................................................................................................................. 18 3.1.1 Consumables ..................................................................................................... 18 3.1.2 Chemicals and reagents..................................................................................... 18 3.1.3 Primers............................................................................................................... 21 3.1.4 Recombinant proteins......................................................................................... 22 3.1.5 Antibodies .......................................................................................................... 23 3.1.6 Enzymes ............................................................................................................ 23 3.1.7 Buffers and other solutions................................................................................. 23 3.1.8 Cell culture media and additives ......................................................................... 27 3.1.9 Kits ..................................................................................................................... 28 3.1.10 Equipment .......................................................................................................... 28 3.1.11 Software and online sources .............................................................................. 30 3.2 Methods ................................................................................................................... 30 3.2.1 Isolation of human mesenchymal stem cells....................................................... 30 3.2.2 Cell culture of human mesenchymal stem cells .................................................. 31 3.2.2.1 Culture and propagation ............................................................................ 31 3.2.2.2 Differentiation............................................................................................. 31 3.2.2.3 Transdifferentiation .................................................................................... 32 Table of contents ii 3.2.2.4 Proliferation assay ..................................................................................... 32 3.2.2.5 Application of recombinant proteins in the cell culture................................ 32 3.2.3 Methods in molecular biology ............................................................................. 34 3.2.3.1 Isolation of cellular RNA............................................................................. 34 3.2.3.2 Reverse transcriptase polymerase chain reaction (RT-PCR) ..................... 34 3.2.3.3 Agarose gel electrophoresis....................................................................... 35 3.2.3.4 DNA sequencing........................................................................................ 35 3.2.3.5 cDNA microarray analysis.......................................................................... 36 3.2.4 Methods in protein analysis ................................................................................ 37 3.2.4.1 Preparation of intracellular protein ............................................................. 37 3.2.4.2 Protein precipitation in culture supernatants .............................................. 38 3.2.4.3 Determination of protein concentration....................................................... 38 3.2.4.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .............................. 38 3.2.4.5 Western blotting......................................................................................... 40 3.2.5 Cytochemical methods ....................................................................................... 40 3.2.5.1 Alizarin red S staining ................................................................................ 40 3.2.5.2 Alkaline phosphatase (ALP) staining.......................................................... 41 3.2.5.3 Oilred O staining ........................................................................................ 41 4 Results.......................................................................................................................... 42 4.1 Adipogenic and osteogenic differentiation potential of human mesenchymal stem cells.......................................................................................................................... 42 4.2 Adipogenic and osteogenic transdifferentiation potential of human mesenchymal stem cells.......................................................................................................................... 44 4.2.1 Adipogenic transdifferentiation............................................................................ 45 4.2.2 Osteogenic transdifferentiation ........................................................................... 47 4.3 Affymetrix microarray analyses of adipogenic and osteogenic transdifferentiation.... 48 4.3.1 Regulation of gene products shortly after initiation of transdifferentiation............ 48 4.3.1.1 Affymetrix GeneChip HG-U 133A............................................................... 48 4.3.1.2 Affymetrix GeneChip HG-U 133 Plus 2.0 ................................................... 49 4.3.2 Re-evaluation of microarray data by semi-quantitative RT-PCR ......................... 52 4.3.3 Gene ontology analyses ..................................................................................... 58 4.3.4 Functional grouping of differentially regulated genes .......................................... 63 4.3.5 Development of a novel bioinformatic scoring scheme for interpretation of microarray results............................................................................................... 65 4.3.5.1 Definition of the criteria for the novel scoring scheme ................................ 65 4.3.5.2 Ranking of differentially regulated gene products by the novel scoring scheme...................................................................................................... 66 Table of contents iii 4.4 Functional testing of human recombinant proteins during differentiation and transdifferentiation.................................................................................................... 70 4.4.1 Secreted proteins tested in differentiation of MSCs without effect....................... 70 4.4.2 Effect of FGF1 on adipogenic differentiation and transdifferentiation .................. 71 5 Discussion...................................................................................................................