MSC isolation, adipogenic differentiation and dedifferentiation.

<p>MSC were induced to adipogenic differentiation for 15 days. (<b>A</b>) Oil Red O staining showed the formation of lipid droplets on day 5, (<b>B</b>) which increased in size and number, as shown on day 10, and (<b>C</b>) reached a peak value on day 15 of adipogenic differentiation. (<b>D</b>) Control samples showed no lipid formation even after day 15 of adipogenesis. Oil Red O staining during the conversion of adipogenic differentiated cells into dedifferentiated cells showed (<b>G</b>) an intermediate conversion after day 7 and (<b>H</b>) complete conversion after day 35 of reverse adipogenesis. Morphology of (<b>F</b>) dedifferentiated cells and (<b>E</b>) undifferentiated MSC are shown by phase contrast microscopy. Bar: 100 µm.</p

Gene expression profile of fat specific marker genes to assess adipogenesis and reverse adipogenesis.

<p>Gene expression analysis was performed using qRT-PCR and the resulted expression data were normalized to <i>GAPDH</i> for stepwise assessment of adipogenesis and reverse adipogenesis. Gene expression of adipogenic-specific marker genes (<b>A</b>) <i>PPARG</i> and (<b>B</b>) <i>FABP4</i> is given for different stages of adipogenic differentiation i.e. at day 5, day 10 and day 15. Similarly, the gene expression of (<b>C</b>) <i>PPARG</i> and (<b>D</b>) <i>FABP4</i> is given for different stages of reverse adipogenesis (dedifferentiation). Error bars, Means ± S.E.M (n = 3); <i>*P</i><0.05; <i>**P</i><0.01; <i>***P</i><0.001, NS, not significant (student t test performed for statistical analysis).</p

Evaluation of different biological parameters for each cluster.

<p>The genes of each cluster were uploaded individually to online databases (DAVID and KEGG) and analyzed for their link to different biological parameters like gene ontology, cellular compartmentalization, molecular function, signaling pathway and site of expression. The parameters were selected on the basis of the enrichment score and relevance for adipogenesis. The numbers given in brackets are the numbers of genes associated to the corresponding GO term and signaling pathways.</p

Reverse Differentiation as a Gene Filtering Tool in Genome Expression Profiling of Adipogenesis for Fat Marker Gene Selection and Their Analysis

<div><p>Background</p><p>During mesenchymal stem cell (MSC) conversion into adipocytes, the adipogenic cocktail consisting of insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine not only induces adipogenic-specific but also genes for non-adipogenic processes. Therefore, not all significantly expressed genes represent adipogenic-specific marker genes. So, our aim was to filter only adipogenic-specific out of all expressed genes. We hypothesize that exclusively adipogenic-specific genes change their expression during adipogenesis, and reverse during dedifferentiation. Thus, MSC were adipogenic differentiated and dedifferentiated.</p><p>Results</p><p>Adipogenesis and reverse adipogenesis was verified by Oil Red O staining and expression of <i>PPARG</i> and <i>FABP4</i>. Based on GeneChips, 991 genes were differentially expressed during adipogenesis and grouped in 4 clusters. According to bioinformatic analysis the relevance of genes with adipogenic-linked biological annotations, expression sites, molecular functions, signaling pathways and transcription factor binding sites was high in cluster 1, including all prominent adipogenic genes like <i>ADIPOQ</i>, <i>C/EBPA</i>, <i>LPL</i>, <i>PPARG</i> and <i>FABP4</i>, moderate in clusters 2–3, and negligible in cluster 4. During reversed adipogenesis, only 782 expressed genes (clusters 1–3) were reverted, including 597 genes not reported for adipogenesis before. We identified <i>APCDD1</i>, <i>CHI3L1</i>, <i>RARRES1</i> and <i>SEMA3G</i> as potential adipogenic-specific genes.</p><p>Conclusion</p><p>The model system of adipogenesis linked to reverse adipogenesis allowed the filtration of 782 adipogenic-specific genes out of total 991 significantly expressed genes. Database analysis of adipogenic-specific biological annotations, transcription factors and signaling pathways further validated and valued our concept, because most of the filtered 782 genes showed affiliation to adipogenesis. Based on this approach, the selected and filtered genes would be potentially important for characterization of adipogenesis and monitoring of clinical translation for soft-tissue regeneration. Moreover, we report 4 new marker genes.</p></div

Isolation of single cells and cultivation of clonal cultures.

<p>Hematoxylin staining of native periosteal tissue (A). Single periosteal cell in cell culture 4 days after enzymatic digestion of the native tissue (B) followed by a separation using cloning cylinders (C,D). Confluent monolayer culture of clonal periosteal cells in passage 1 at day 5 (E) and in passage 9 at day 7 (F); A, B, E, F: 100x magnification.</p

New potential fat marker genes, selected based on the coupling model of adipogenesis and reverse adipogenesis.

<p>Gene expression analysis was performed using qRT-PCR and the expression values were normalized to <i>GAPDH</i> for stepwise assessment of adipogenesis and reverse adipogenesis (dedifferentiation). Gene expression of new potential fat marker genes (<b>A</b>) <i>APCDD1</i>, (<b>B</b>) <i>SEMA3G</i>, (<b>C</b>) <i>CHI3L1</i> and (<b>D</b>) <i>RARRES1</i> is given for different stages of adipogenesis, i.e. at day 5, day 10 and day 15. Similarly, the expression of (<b>E</b>) <i>APCDD1</i>, (<b>F</b>) <i>SEMA3G</i>, (<b>G</b>) <i>CHI3L1</i> and (<b>H</b>) <i>RARRES1</i> is given for different stages of dedifferentiation (reverse adipogenesis). Here the gene expression of adipogenic differentiated cells is represented by day 0 as a reference for dedifferentiation. Error bars, Means ± S.E.M (n = 3); <i>*P</i><0.05; <i>**P</i><0.01; <i>***P</i><0.001, NS, not significant (student t test, performed for statistical analysis).</p

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application

<div><p>For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR) of <i>BGLAP</i> and <i>RUNX2</i> and for adipogenesis of <i>FABP4</i> and <i>PPARG</i> confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation), 3.9% bipotent (without chondrogenic differentiation), and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them <i>DCN</i>, <i>NEDD9</i>, <i>TGFBR3</i>, and <i>TSLP</i>. For clinical applications of periosteal cells in bone regeneration all cells were inducible. For a chondrogenic application a fraction of 6% of the mixed population could not be induced.</p></div

Transcription factor binding sites (TFBS) analysis.

<p>Analysis of transcription factor binding sites (TFBS) was performed and the selected adipogenic-specific TFBS showed most of the binding sites in cluster 1–3 genes and only a few significant sites in cluster 4 genes.</p

Classification of clonal cultures according to the maximal passage number P_max and the mean growth rate μ.

<p>Classification of clonal cultures according to the maximal passage number P_max and the mean growth rate μ.</p

Distribution of clonal cultures of the 4 donors according to their growth classes.

<p>Distribution of clonal cultures of the 4 donors according to their growth classes.</p