A mesenchymal stromal cell gene signature for therapeutic efficacy of autologous bone tissue engineering
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1 A mesenchymal stromal cell gene signature for therapeutic efficacy of autologous bone tissue engineering Jan de Boer University of Twente MIRA Institute for Biomedical Technology Department of Tissue Regeneration Enschede, The Netherlands
2 Bone tissue engineering expansion Non-union fractures Resection of bone tumors Wear of implants osteoblast Spinal fusion + surgery High bone energy graft trauma MSC substitute differentiation scaffold Petite et al., Nature Biotech, 2000 Meijer et al., PLOS Medicine 2007
3 Animal MSCs outperform human MSCs in ectopic bone formation Rat Human Siddappa et al., Cur Stem Cell Res, 2007
4 Donor variability in the osteogenic respons No strong correlation to ectopic bone formation Siddappa et al, J Orth Res, 2007
5 MSC CD typing 60% 0,3% CD % CD 11b 0% Other markers: Stro-1 NGFR CD 73 97% CD 34 0% Relation to CFU-F, not in vivo bone CD 90 CD 19 0% CD 45 1,7% HLA-DR CD146 Haematopoietic microenvironment
6 Johan Cruijf: elk nadeel heb z n voordeel every drawback has its advantage Use donor variability to our benefit in order to identify: 1. diagnostic markers predictive for bone formation in vivo 2. osteogenic MSC subpopulations 3. signal transduction routes critical for bone formation by MSCs in vivo
7 Systematic approach to find a correlation between molecular markers in expanding hmscs and bone apposition in vivo Clinical characteristics Growth and differentiation characteristics 62 donors In vivo bone Microarray
8 Relative Adipogenesis (OD) Frequency Heterogeneity in vitro labels PD/day P0-P Donor number
9 % Bone area / scaffold area Heterogeneity in ectopic bone formation Donor number 35 bone forming donors, 27 non-bone formers
10 % Bone area / scaffold area No correlation between ALP expression and bone formation % ALP positive cells (dex)
11 Diagnostic classifiers for bone label prediction ROC= receiver operating curve AUC= area under curve
12 Correlating gene expression profiles with labels Site of aspiration Homeotic genes Clean dataset
13 CADM1 expression is highly predictive for bone formation by hmscs B Mentink et al, submitted
14 CadM1 a.k.a. BL2; DKFZp686F1789; IGSF4; IGSF4A; MGC149785; MGC51880; NECL2; Necl-2; RA175; ST17; SYNCAM; TSLC1; stslc-1; syncam1 Reported roles: Neuronal synapse formation Tumor suppressor gene Heterotypic cell/cell interaction e.g.sperm/sertoli and mast cell/smooth muscle cell Not reported in relation to MSCs or bone
15 CaDM1 immunostaining GLC4 hmsc
16 Relative Calcium Accumulation (%) CadM1 expression decreases upon MSC expansion * * * * * * *** *** *** RPD 6 RPD 10 RPD 13 RPD 17 RPD 20 RPD 25 CadM1 expression mineralisation Alves et al., JCMM 2009
17 Top 50 of genes correlating to bone formation Neuronal signature CADM1 Gamma-aminobutyric acid B receptor 2 Semaphorin 5A Glycoprotein M6B Carboxypeptidase E Wnt signature WISP1 WISP2 Dickkopf-1 S100A4 IGF signaling signature IGF1 IGF2 SocS2
18 CadM expression is induced by Wnt agonist GIN 35 CADM1 expression D.234 D.234+GIN
19 Conclusions: Large donor heterogeneity exists with respect to bone formation Traditional markers do not robustly predict bone formation A single gene is highly predictive and may be used as diagnostic screen for inclusion of donors in bone TE tool to identify an MSC subpopulation with bone-forming capacity molecular marker to screen reagents or biomaterials which favor the osteogenic potential of MSCs lead to novel insight in the role of MSCs in bone formation
20 Acknowledgements Twente University Anouk Mentink Ruud Licht Clemens van Blitterswijk Organon N.V. Koen Dechering Anja Garritsen Technical University Delft Marcel Reinders Eugene van Someren Marc Hulsman UMC Utrecht Wouter Dhert Laura Creemers MST Hospital Enschede Auke Renard Istvan Vermes Funding Senter/Novem DPTE Smartmix
21 AUC scores of probe sets for bone classifiers
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