Identification of Markers to Characterize and Sort Human Articular Chondrocytes With Enhanced In Vitro Chondrogenic Capacity

Grogan, Shawn Patrick
Barbero, Andrea
Diaz-Romero, Jose
Cleton-Jansen, Anne-Marie
Soeder, Stephan
Whiteside, Robert
Hogendoorn, Pancras C. W.
Farhadi, Jian
Aigner, Thomas
Martin, Ivan
Mainil-Varlet, Pierre

¹University of Bern, Bern, Switzerland
²University Hospital Basel, Basel, Switzerland
³Leiden University Medical Center, Leiden, The Netherlands
⁴Institute of Pathology, Leipzig, Germany

Address correspondence and reprint requests to Ivan Martin, Institute for Surgical Research and Hospital Management, University Hospital Basel, Hebelstrasse 20, ZLF, Room 405, 4031 Basel, Switzerland. E-mail: [email protected]

(current address: The Scripps Research Insitute, La Jolla, California

Drs. Grogan and Barbero contributed equally to this work.

Supported by grants from the Swiss National Science Foundation (SNF 4046-58623) and the Swiss Commission for Technology and Innovation (CTI 6312.1). Dr. Martin is recipient of an educational grant from Millenium Biologix.

Submitted for publication August 11, 2006; accepted in revised form November 09, 2006

Arthritis & Rheumatism 56(2):p 586-595, February 2007.

Objective

To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations.

Methods

The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures.

Results

Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell–cell or cell–matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells.

Conclusion

We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.