Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors
Kunihiko Hiramatsu, Satoru Sasagawa, Hidetatsu Outani, Kanako Nakagawa, Hideki Yoshikawa, Noriyuki Tsumaki
Kunihiko Hiramatsu, Satoru Sasagawa, Hidetatsu Outani, Kanako Nakagawa, Hideki Yoshikawa, Noriyuki Tsumaki
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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 4

Chondrocyte marker gene expression analyses of induced cell lines.

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Chondrocyte marker gene expression analyses of induced cell lines. (A) R...
(A) Real-time RT-PCR analysis of chondrocyte marker gene expression in MK induced cells, MDFs, and primary chondrocytes. Error bars indicate mean ± SD (n= 3). (B) Real-time RT-PCR analysis of fibroblast marker gene expression in MK induced cells, MDFs, and primary chondrocytes. MK-4 cells expresseda small amount of Col1a1. Error bars indicate mean ± SD (n= 3). (C) Immunofluorescence staining showing expression of marker proteins (red) in MK-4 and -5 cells, MDFs, and primary chondrocytes. Blue nuclear staining is DAPI. Scale bar: 100 μm. (D) Bisulfite genomic sequencing of the promoter regions of Col1a1 and Col1a2. Each horizontal row of circles represents an individual sequencing result from 1 amplicon. Open circles indicate unmethylated CpG dinucleotides, while closed circles indicate methylated CpGs. (E) Immunofluorescence analysis showing protein expression (red) of SOX9/Sox9, Klf4, and c-Myc in MK-4 and MK-5 cells and expression of Sox9 in primary chondrocytes. Blue nuclear staining is DAPI. Scale bar: 100 μm. (F) Transgenic SOX9 mRNA levels and endogenous Sox9 mRNA levels were determined by real-time RT-PCR analysis with primers specific for transgenic SOX9 transcripts and for endogenous Sox9 transcripts, respectively. Shown are individual RNA expression levels normalized to respective Gapdh expression levels. Error bars indicate mean ± SD (n= 3). Hy Ch, hypertrophic chondrocyte zone of epiphyseal cartilage from neonatal femur and tibia.