Dual luciferase labelling for non-invasive bioluminescence imaging of mesenchymal stromal cell chondrogenic differentiation in demineralized bone matrix scaffolds Marta Vilalta a , Christian Jorgensen b, c , Irene R. De ´ gano a , Yuti Chernajovsky d , David Gould d , Danie ` le Noe ¨l b, c , Jose ´ A. Andrades e , Jose ´ Becerra e , Nuria Rubio a , Jero ´ nimo Blanco a, * a Cardiovascular Research Center, CSIC-ICCC, CIBER-BBN, Barcelona 08025, Spain b INSERM U 844, Montpellier F-34091, France c Universite´ MONTPELLIER1, UFR de Me´decine, Montpellier F-34000, France d Bone and Joint Research Unit, Barts and The London, Queen Mary’s School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK e Department of Cell Biology, Genetics and Physiology, Laboratory of Bioengineering and Tissue Regeneration, Faculty of Sciences, University of Ma ´laga, CIBER-BBN, Ma ´laga, Spain article info Article history: Received 23 March 2009 Accepted 21 May 2009 Available online 17 June 2009 Keywords: Bioluminescence imaging (BLI) Collagen type II (COL2A1) Demineralized bone matrix (DBM) Human adipose tissue derived mesenchymal stromal cells (hAMSCs) Murine cell line (CL1) abstract Non-invasive bioluminescence imaging (BLI) to monitor changes in gene expression of cells implanted in live animals should facilitate the development of biomaterial scaffolds for tissue regeneration. We show that, in vitro, induction of chondrogenic differentiation in mouse bone marrow stromal cell line (CL1) and human adipose tissue derived mesenchymal stromal cells (hAMSCs), permanently transduced with a procollagen II (COL2A1) promoter driving a firefly luciferase gene reporter (PLuc) (COL2A1p$PLuc), induces PLuc expression in correlation with increases in COL2A1 and Sox9 mRNA expression and acquisition of chondrocytic phenotype. To be able to simultaneously monitor in vivo cell differentiation and proliferation, COL2A1p$PLuc labelled cells were also genetically labelled with a renilla luciferase (RLuc) gene driven by a constitutively active cytomegalovirus promoter, and then seeded in deminer- alized bone matrix (DBM) subcutaneously implanted in SCID mice. Non-invasive BLI monitoring of the implanted mice showed that the PLuc/RLuc ratio reports on gene expression changes indicative of cell differentiation. Large (CL1) and moderated (hAMSCs) changes in the PLuc/RLuc ratio over a 6 week period, revealed different patterns of in vivo chondrogenic differentiation for the CL1 cell line and primary MSCs, in agreement with in vitro published data and our results from histological analysis of DBM sections. This double bioluminescence labelling strategy together with BLI imaging to analyze behaviour of cells implanted in live animals should facilitate the development of progenitor cell/scaffold combinations for tissue repair. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Non-invasive bioluminescent imaging (BLI) could provide additional insight to that generated by more conventional in vitro approaches, to understand cell proliferation and differentiation changes taking place in live animals during normal tissue devel- opment or cell therapy. As previously described, the proliferation and distribution of cells labelled with bioluminescent reporters can be monitored by BLI, during extended time periods after being directly seeded in scaffolds and implanted in live animals for therapy [1–3]. Thus, during development of scaffolds for tissue regeneration, it would be particularly useful to non-invasively monitor, in real time, the behaviour of cell–scaffold combinations implanted in live animals. Due to the capacity of light photons to transverse living tissues, BLI of photoprotein gene reporters introduced in cells can be used to monitor biodistribution and gene expression in vivo. Thus, by using tissue specific gene promoters to regulate bioluminescent reporters, changes in promoter activity induce changes in reporter expression detectable as photon fluxes. Analysis of photon images provides information on cell distribution and time related gene expression changes associated to cell differentiation stages [4–6]. * Corresponding author. Cardiovascular Research Center, CSIC-ICCC, CIBER-BBN, C/ Sant Atnoni M a Claret, 167, Barcelona 08025, Spain. Tel.: þ34 935565905; fax: þ34 93556555. E-mail address: jblancof@csic-iccc.org (J. Blanco). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.05.056 Biomaterials 30 (2009) 4986–4995