Dual luciferase labelling for non-invasive bioluminescence imaging of mesenchymal stromal cell chondrogenic differentiation in demineralized bone matrix scaffolds.
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 demineralized 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.
Vilalta
Marta
M
Cardiovascular Research Center, CSIC-ICCC, CIBER-BBN, Barcelona 08025, Spain.
Jorgensen
Christian
C
Dégano
Irene R
IR
Chernajovsky
Yuti
Y
Gould
David
D
Noël
Danièle
D
Andrades
José A
JA
Becerra
José
J
Rubio
Nuria
N
Blanco
Jerónimo
J
D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology; D024043:Collagen Type II / Q000235:genetics* / Q000378:metabolism; D003952:Diagnostic Imaging / Q000379:methods; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D010012:Osteogenesis; D017154:Stromal Cells / Q000166:cytology; D054457:Tissue Scaffolds; D014161:Transduction, Genetic
C499177:COL2A1 protein, human; D024043:Collagen Type II; D049409:Luciferases, Firefly