Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors
Kunihiko Hiramatsu, … , Hideki Yoshikawa, Noriyuki Tsumaki
Kunihiko Hiramatsu, … , Hideki Yoshikawa, Noriyuki Tsumaki
Published January 10, 2011
Citation Information: J Clin Invest. 2011;121(2):640-657. https://doi.org/10.1172/JCI44605.
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Technical Advance

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

Abstract

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

Authors

Kunihiko Hiramatsu, Satoru Sasagawa, Hidetatsu Outani, Kanako Nakagawa, Hideki Yoshikawa, Noriyuki Tsumaki

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Figure 9

Analysis of induced cells from Col11a2-Egfp-Ires-Puro transgenic MDFs by dox-inducible lentiviral c-MYC, KLF4, and SOX9 vectors.

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Analysis of induced cells from Col11a2-Egfp-Ires-Puro transgenic MDFs by...
(A) Real-time RT-PCR analysis of the dox-inducible transgenes in induced cell lines (nos. 290-2-14 and 290-2-19) cultured in DMEM + 10% FBS in the presence or absence of dox. Relative expression levels in cells cultured in the presence of dox are set as 1. (B) Growth analysis of induced cell lines (nos. 290-2-6, 290-2-14, and 290-2-19) cultured in DMEM plus 10% FBS in the presence or absence of dox. A total of 1 × 105 induced cells were seeded in 6-well plates, and cell numbers were determined every other day. (C) Toluidine blue staining of induced cells and MDFs. Induced cells were cultured in DMEM PLUS 10% FBS in the presence (top left) or absence (top right) of dox for 14 days. The culture medium of induced cells was changed to chondrogenic medium with dox (middle left panel) or chondrogenic medium without dox (middle right panel), and cells were cultured for 7 days. (D) Phase and GFP images of induced cells cultured in chondrogenic medium in the presence or absence of dox for 7 days. Scale bar: 100 μm. (E) Real-time RT-PCR analysis of induced cells. Culture media of induced cells was changed from DMEM plus 10% FBS and dox to the chondrogenic medium with dox (+) or chondrogenic medium without dox (–). After 3 days, total RNAs were extracted. Error bars indicate mean ± SD (n= 3).