Temporal Patterns of Expression of Type II Procollagen Splice Forms During the Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Yoo, Jeong Joon; Kim, Jong Won; Kim, Young Min; Kim, Hee Joong

Abstract

PURPOSE
We investigated the temporal patterns of expression of alternatively spliced Type II procollagen mRNAs during the chondrogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) in alginate bead and pellet culture systems.
MATERIALS AND METHODS
hMSCs were prepared from bone marrow aspirates taken from the iliac crest of six normal human donors and expanded in monolayer culture. After 4 passages, the expanded cells were induced to chondrogenic differentiation with 10 ng/ml rhTGF-beta3 and cultured for 28 days in two different three dimensional culture systems, alginate bead culture and pellet culture. Reverse transcription-polymerase chain reaction (RT-PCR) for the expression of alternatively spliced type II procollagen mRNAs was performed with three sets of primers during the culture periods. Immunohistochemical staining (IHC) for type II collagen and glycosaminoglycan content assay were done to confirm the commitment of hMSCs to chondrogenic lineage.
RESULTS
RT-PCR revealed that the expression of type II and type IIA procollagen mRNA increased gradually during chondrogenic differentiation in both culture systems. When both type IIA and IIB procollagen transcripts were amplified at the same time, it was found that the alternative splicing of type II procollagen mRNAs does occur and the expression levels of both forms increased gradually during the chondrogenic differentiation. In the undifferentiated cells, none of them was detected. During the chondrogenic differentiation of hMSCs, type II collagen was detected with IHC staining and glycosaminoglycan content increased gradually.
CONCLUSION
In this study, it was confirmed that the alternative splicing of type II procollagen gene does occur and type IIA procollagen mRNA increased gradually during the chondrogenic differentiation. The present results indicate that the expression levels of type II procollagen splice forms (type IIA and IIB) should be evaluated simultaneously for the better assessment of chondrogenic differentiation of hMSCs and the chondrogenic differentitation of hMSCs in alginate bead or pellet culture system can be used as an in vitro model for the research on the biological processes of type II procollagen.