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Characterization of long‐term in vitro culture‐related alterations of human tonsil‐derived mesenchymal stem cells: role for CCN 1 in replicative senescence‐associated increase in osteogenic differentiation
Author(s) -
Yu Yeonsil,
Park Yoon Shin,
Kim Han Su,
Kim Ha Yeong,
Jin Yoon Mi,
Jung SungChul,
Ryu KyungHa,
Jo Inho
Publication year - 2014
Publication title -
journal of anatomy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 118
eISSN - 1469-7580
pISSN - 0021-8782
DOI - 10.1111/joa.12229
Subject(s) - mesenchymal stem cell , cd90 , stem cell , homeobox protein nanog , cd146 , cellular differentiation , microbiology and biotechnology , biology , cd34 , oil red o , embryonic stem cell , adipogenesis , cd44 , chemistry , induced pluripotent stem cell , cell , biochemistry , gene
Although mesenchymal stem cells ( MSC ) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture‐related alterations in their stem cell properties, such data have not been reported in human tonsil‐derived MSC ( T ‐ MSC ). Here, we investigated the culture‐related changes of phenotypes, the senescence, and the differentiation potential of T ‐ MSC . T ‐ MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC ‐specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC ‐specific surface marker, CD 14, CD 34, CD 45, CD 73 and CD 90, or the m RNA expression of embryonic stem cell gene markers, N anog , O ct4‐ A and S ox‐2 . However, the expression of CD 146, recently identified another MSC marker, dramatically decreased with increasing passages from ~ 23% at passage 3 to ~ 1% at passage 15. The average doubling time increased significantly from ~ 38 h at passage 10 to ~ 46 h at passage 15. From passage 10, the cell size increased slightly and SA ‐β‐gal staining was evident. Both A lizarin R ed S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage‐dependently from the start, as evidenced by staining of O il R ed O and A lcian Blue, respectively. Consistent with a passage‐dependent osteogenic differentiation, the expression of CCN 1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN 1 at passage 10 significantly reversed A lizarin R ed S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long‐term in vitro cultured T ‐ MSC and that CCN 1 may be involved in mediating a passage‐dependent increase in osteogenic potential of T ‐ MSC .