Microarray Analyses of Transdifferentiated Mesenchymal Stem Cells
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Journal of Cellular Biochemistry 103:413–433 (2008) ARTICLES Microarray Analyses of Transdifferentiated Mesenchymal Stem Cells Tatjana Schilling,1 Robert Ku¨ffner,2 Ludger Klein-Hitpass,3 Ralf Zimmer,2 Franz Jakob,1 and Norbert Schu¨tze1* 1University of Wu¨rzburg, Orthopedic Department, Orthopedic Center for Musculoskeletal Research, Wu¨rzburg, Germany 2Ludwig Maximilians University of Munich, Department of Informatics, Bioinformatics, Munich, Germany 3University of Duisburg-Essen, Institute of Cell Biology (Tumor Research), Essen, Germany Abstract The molecular events associated with the age-related gain of fatty tissue in human bone marrow are still largely unknown. Besides enhanced adipogenic differentiation of mesenchymal stem cells (MSCs), transdifferentiation of osteoblast progenitors may contribute to bone-related diseases like osteopenia. Transdifferentiation of MSC-derived osteoblast progenitors into adipocytes and vice versa has previously been proven feasible in our cell culture system. Here, we focus on mRNA species that are regulated during transdifferentiation and represent possible control factors for the initiation of transdifferentiation. Microarray analyses comparing transdifferentiated cells with normally differentiated cells exhibited large numbers of reproducibly regulated genes for both, adipogenic and osteogenic transdifferentiation. To evaluate the relevance of individual genes, we designed a scoring scheme to rank genes according to reproducibility, regulation level, and reciprocity between the different transdifferentiation directions. Thereby, members of several signaling pathways like FGF, IGF, and Wnt signaling showed explicitly differential expression patterns. Additional bioinformatic analysis of microarray analyses allowed us to identify potential key factors associated with trans- differentiation of adipocytes and osteoblasts, respectively. Fibroblast growth factor 1 (FGF1) was scored as one of several lead candidate gene products to modulate the transdifferentiation process and is shown here to exert inhibitory effects on adipogenic commitment and differentiation. J. Cell. Biochem. 103: 413–433, 2008. ß 2007 Wiley-Liss, Inc. Key words: transdifferentiation; mesenchymal stem cells; osteoblast; adipocyte; microarray analysis Maintenance and regeneration of bone re- et al., 1999; Muraglia et al., 2000; Verfaillie, quires the differentiation of human mesenchy- 2002; No¨th et al., 2002a; Barry and Murphy, mal stem cells (hMSCs) into osteoblasts [Krane, 2004]. Since adipogenic degeneration in the 2005; Martin and Sims, 2005]. Besides genera- bone marrow and bone-associated diseases like tion of osteoblasts, the multipotential hMSCs osteoporosis and osteopenia have been reported can also generate adipocytes and other mesen- to increase with aging, an inverse relationship chymal cell lineages [Prockop, 1997; Pittenger between osteogenic and adipogenic differentia- tion of hMSCs has been assumed [Meunier et al., 1971; Burkhardt et al., 1987; Beresford et al., 1992; Koo et al., 1998; Nuttall and Gimble, This article contains supplementary material, which may 2000; Pei and Tontonoz, 2004; Gimble et al., be viewed at the Journal of Cellular Biochemistry website 2006; Rosen and Bouxsein, 2006]. Further at http://www.interscience.wiley.com/jpages/0730-2312/ evidence for this inverse relationship was ob- suppmat/index.html. tained in a mouse model, where over-expression Grant sponsor: Deutsche Forschungsgemeinschaft; Grant of 12/15-lipoxygenase that indirectly stimulates number: SCHU 747/7-1. adipogenesis caused osteopenia in these ani- *Correspondence to: Norbert Schu¨ tze, University of Wu¨ rz- mals [Klein et al., 2004]. Thus, a reduction of the burg, Orthopedic Department, Orthopedic Center for Musculoskeletal Research, Molecular Orthopedics, Brett- capability of hMSCs to differentiate into osteo- reichstr. 11, D-97074 Wu¨ rzburg, Germany. blasts thereby favoring adipogenesis as well as E-mail: [email protected] transdifferentiation of committed osteoblasts Received 21 December 2006; Accepted 18 April 2007 into adipocytes could contribute to the adipo- DOI 10.1002/jcb.21415 genic degeneration. ß 2007 Wiley-Liss, Inc. 414 Schilling et al. Transdifferentiation occurs when a committed Thereby, functional examination of fibroblast cell type that already develops along a certain growth factor 1 (FGF1) as one of the potential differentiation pathway (e.g., a pre-osteoblast) key factors was proven to inhibit adipogenic changes into a cell type of another differentiation lineage development. lineage (e.g., an adipocyte). Such phenotype switches between committed or differentiated MATERIALS AND METHODS osteoblasts and adipocytes have been reported by us and others [Nuttall et al., 1998; Park et al., Chemicals were obtained from Sigma– 1999; Schiller et al., 2001; Schilling et al., 2007], Aldrich (Munich, Germany) unless specified even at the single cell level [Song and Tuan, otherwise. Cell culture media were purchased 2004]. The signaling molecules and pathways from PAA (Co¨lbe, Germany), fetal bovine serum leading to adipogenic or osteogenic transdiffer- (FBS) from Gibco (Karlsruhe, Germany) or PAA entiation are completely unknown. If and to (Co¨lbe, Germany). what extent established key factors and signal- Isolation and Culture Expansion of hMSCs ing pathways of normal osteogenesis and adipo- genesis are involved in the reprogramming hMSCs from trabecular bone were isolated process is also unknown so far. from the femoral heads of patients undergoing Osteogenesis is induced by runt-related tran- hip replacement surgery due to age-related or scription factor 2 (RUNX2) and osterix [Ducy hip dysplasia-related attrition and cultivated as et al., 1997; Ducy, 2000; Nakashima et al., described previously [Haynesworth et al., 1992; 2002; Nakashima and de Crombrugghe, 2003]. No¨th et al., 2002b; Schu¨ tze et al., 2005a]. It is further enhanced via extracellular signal- Patients were otherwise healthy and did not regulated kinases 1 and 2 involved in the receive medications with relation to bone meta- mitogen-activated protein kinase pathway bolism. Experiments were performed upon [Jaiswal et al., 2000; Xiao et al., 2002], by bone approval by the Local Ethics Committee of the morphogenetic protein 2, and D FBJ murine University of Wu¨ rzburg and informed consent osteosarcoma viral oncogene homolog B (D from each patient (seven female patients aged FOSB) [Gori et al., 1999; Sabatakos et al., 40–75 years and one male patient aged 2000]. Adipogenesis is induced by the sequen- 49 years). Cells were propagated in expansion tial action of C/CAAT enhancer binding proteins medium consisting of DMEM/Ham’s F-12 (1:1) (CEBPs) and peroxisome proliferator-activated with L-glutamine supplemented with 10% heat receptor g 2 (PPARG2) [Tanaka et al., 1997; inactivated FBS, 1 U/ml penicillin, 100 mg/ml Darlington et al., 1998; Wu et al., 1999; Rosen streptomycin (PAA), and 50 mg/ml L-ascorbic et al., 2000] that results in enhanced expression acid 2-phosphate. Cells were plated for (trans-) of other adipocyte-specific genes like acetyl CoA differentiation experiments in passage 1 or 2. carboxylases and fatty acid binding protein 4 Differentiation and Transdifferentiation (FABP4) [Spiegelman et al., 1993]. of hMSCs Transdifferentiation of hMSC-derived osteo- blast progenitors and adipocytes into adipocytes Differentiation and transdifferentiation of and osteoblasts, respectively, was demon- hMSCs were performed as described previously strated in our previous work [Schilling et al., [Schilling et al., 2007]. Briefly, osteogenic 2007]. Since the molecular mechanisms provok- differentiation of hMSCs was achieved by ing this reprogramming are unknown, we incubation of confluent hMSC monolayers in analyzed genome-wide gene expression pat- osteogenic medium (OM) consisting of expan- terns by employment of an Affymetrix Gene- sion medium additionally supplemented with Chip representing 38,500 genes and by 10 mM b-glycerophosphate and 100 nM dex- development of a novel scoring scheme that amethasone for up to 4 weeks [Jaiswal et al., evaluates the relevance of regulated genes in 1997; Schu¨ tze et al., 2005b]. Confluent hMSCs the processes of transdifferentiation. Our find- formed adipocytes after exposure to adipogenic ings serve as prerequisite for functional exam- medium (AM) consisting of DMEM high glucose inations of promising candidates that could be with L-glutamine supplemented with 10% heat- involved in signaling pathways provoking inactivated FBS, 1 U/ml penicillin, 100 mg/ml transdifferentiation or even acting as switches streptomycin, 1 mM dexamethasone, 100 mM between the adipogenic and osteogenic lineage. indomethacin, 500 mM 3-isobutyl-1-methyl- Microarray Analyses of Transdifferentiation 415 xanthine, and 1 mg/ml insulin after 2 weeks affymetrix.com). In short, biotinylated cRNA [Pittenger et al., 1999; Schu¨ tze et al., 2005b]. samples were prepared from total RNA Transdifferentiation of committed osteoblasts samples and hybridized onto GeneChips con- (14 days in OM) was performed by subsequent taining probe sets for 47,400 transcripts incubation in AM for another 14 days. Analo- and 38,500 genes, respectively (http://www. gously, fully differentiated adipocytes (14 days in affymetrix.com/support/technical/datasheets/ AM) were transdifferentiated into osteoblasts by human_datasheet.pdf). Hybridization signals exposure to OM for another 4 weeks. detected with the Affymetrix GeneChip Confluent hMSCs, plated at a density