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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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 3

Characterization of induced cell lines.

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Characterization of induced cell lines. (A) Growth curves of induced cel...
(A) Growth curves of induced cell lines and MDFs. A total of 1 × 105 cells were passaged every 3 days into each well of 6-well plates. Averages of 4 separate experiments are shown. (B) Southern blot analysis of genomic DNA extracted from MK lines and MDFs. Genomic DNA was digested with EcoRI and BamHI, separated on an agarose gel, transferred to a nylon membrane, and hybridized with Klf4 cDNA probe. (C) Karyotype of the MK-5 cell line. (D) When the calculated cell numbers in the growth curve exceeded 1010, the cells were replated on 10-cm dishes. MDF culture (passage 3) was used for a control. After reaching confluence, induced chondrogenic cells and MDFs were cultured for an additional 14 days and stained with alcian blue. (E) Cell morphologies of induced cell lines. Phase contrast images are shown. Scale bar: 100 μm. (F) Pellet culture of MK-7 and MK-10 cells. After 3 weeks of culture, pellets were recovered and processed for histological sections. Semiserial sections were stained with safranin O-fast green-iron hematoxylin and toluidine blue, and immunostained with anti–type II collagen antibodies, anti–type I collagen antibodies, and anti–type X collagen antibodies. Scale bar: 50 μm.