Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Analysis of Secretome from Mesenchymal Stem Cells from Human Umbilical Cord Strome during the Chondrogenesis.
Arufe1, María C., De la Fuente2, Alexandre, Mateos4, Jesús, Fernandez-Puente4, Patricia, Rendal4, Esther, Diaz2, Silvia, Fuentes2, Isaac
INIBIC-Universidad de A Coruna, A Coruna, Osteoarticular and Aging Research Laboratory, Proteomics Unit-Associated Node to ProteoRed-Biomedical Research Center (INIBIC), Hospital Universitario A Coruna, La Coruna, Coruña, Sp
INIBIC-Universidad de A Coruna, A Coruna, Spain
INIBIC-Universidad de A Coruna, A Coruna, Spain. Osteoarticular and Aging Research Laboratory, Proteomics Unit-Associated Node to ProteoRed-Biomedical Research Center (INIBIC). Hospital Universitario A Coruna, La Coruna, Spain
Osteoarticular and Aging Research Laboratory, Proteomics Unit-Associated Node to ProteoRed-Biomedical Research Center (INIBIC), Hospital Universitario A Coruña. La Coruña, Spain
Our goal is study the secreted proteins from Mesenchymal Stem Cells MSCs- from umbilical cord stroma during their differentiation towards chondrocyte-like cells to discern the pathways which could be determinant in this chodrogenesis.
Material and Methods:
Umbilical cord tissues were obtained from caesareans from normal women in the Maternity Facility at Complejo Hospitalario Universitario de A Coruña under the supervision of the hospital ethic committee. We isolated and growth MSCs from umbilical cord stromal tissue by enzymatic digestion and cultured the cells adhered to the plastic plate(1). The cells were characterized (2) using flow cytometry, immunohistochemistry and RT-PCR techniques. Chondrogenic process was performed using our previously published model (3). Briefly, we grow the cells during two days in medium suplemented with FCS 10%. After 2 days we washed the cells with PBS and added medium supplemented with KO serum and TGF-b3. Spheroids were formed by two days in culture and this three-dimensional structure help to produce the characteristic proteins which form part of the extracellular matrix of the cartilage. The spheroid were situated in a well with the chondrogenic factors plus RPMI during 12 h to recover the protein secreted to the medium by the spheroids at 4, 7, 14, 28 and 46 days of differentiation avoiding the serum contamination.
SDS-PAGE was done using pre-cast NuPAGE® NOVEX gels (Invitrogen) to ensure reproducibility in the separation of the proteins. Gels were stained with Silver Nitrate and the entire lane was divided in 16 sections. Each section was excised and subjected to in-gel digestion with trypsin. The peptide mixture was cleaned and desalted with home-made Poros R2 columns, injected and separated in a nanoLC system, mixed with a-cyano and deposited in a MALDI plate using an automatic MALDI spotter. The MS run for each fraction was analyzed in an ABI 4800 MALDI-TOF/TOF instrument and fragmentation was done using a CID system.
87 identified proteins were related with the chondrogenic differentiation process from a total of 209 proteins. Some of the proteins related with chondrogenesis obtained with this method were:
|Name||SwissProt Accession #||Type||Day|
|Collagen alpha-1||P02452||Collagen||4, 7|
|Collagen alpha-2||P08123||Collagen||4, 7|
|Collagen alpha-3||P12111||Collagen||14, 28|
|Lumican||P51884||Proteoglycan||7, 14, 28|
1. This technique is suitable to analyze medium complexity samples as secretome.
2. The chondrogenesis differentiation designed through spheroid formation have revealed be an excelent method to obtain chondrocytes-like cells from mesenchymal stem cells of human umbilical cord stromal tissue.
3. Pathways related with differentiation process are been activated during the chondrogenesis process.
To cite this abstract, please use the following information:
Arufe, María C., De la Fuente, Alexandre, Mateos, Jesús, Fernandez-Puente, Patricia, Rendal, Esther, Diaz, Silvia, et al; Analysis of Secretome from Mesenchymal Stem Cells from Human Umbilical Cord Strome during the Chondrogenesis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1473