Archives

  • 2025-11
  • 2025-10
  • 2025-09
  • 2025-03
  • 2025-02
  • 2025-01
  • 2024-12
  • 2024-11
  • 2024-10
  • 2024-09
  • 2024-08
  • 2024-07
  • 2024-06
  • 2024-05
  • 2024-04
  • 2024-03
  • 2024-02
  • 2024-01
  • 2023-12
  • 2023-11
  • 2023-10
  • 2023-09
  • 2023-08
  • 2023-07
  • 2023-06
  • 2023-05
  • 2023-04
  • 2023-03
  • 2023-02
  • 2023-01
  • 2022-12
  • 2022-11
  • 2022-10
  • 2022-09
  • 2022-08
  • 2022-07
  • 2022-06
  • 2022-05
  • 2022-04
  • 2022-03
  • 2022-02
  • 2022-01
  • 2021-12
  • 2021-11
  • 2021-10
  • 2021-09
  • 2021-08
  • 2021-07
  • 2021-06
  • 2021-05
  • 2021-04
  • 2021-03
  • 2021-02
  • 2021-01
  • 2020-12
  • 2020-11
  • 2020-10
  • 2020-09
  • 2020-08
  • 2020-07
  • 2020-06
  • 2020-05
  • 2020-04
  • 2020-03
  • 2020-02
  • 2020-01
  • 2019-12
  • 2019-11
  • 2019-10
  • 2019-09
  • 2019-08
  • 2019-07
  • 2019-06
  • 2019-05
  • 2019-04
  • 2018-11
  • 2018-10
  • 2018-07

    • We recently reported in vitro induction of HC like cells

    2018-10-20

    We recently reported in vitro induction of HC-like crf hormone from mouse ES cells using ST2 stromal cell conditioned medium (ST2-CM), termed the HIST2 method, which consists of only two steps, embryoid body (EB) formation for 4days and subsequent EB outgrowth culture for 14days in ST2-CM (Ouji et al., 2012). That method induced approximately 20% of established EB outgrowths to produce HC-like cells. In addition, we reported that ES cells carrying Tet-inducible Math1 (Math1-ES cells) showed a high ability to differentiate into HC-like cells even in unconditioned normal medium, as those comprised approximately 10% of the cells in EB outgrowths grown in the presence of doxycycline (Dox) (Ouji et al., 2013). In the present study, we used the HIST2 method in combination with Math1-ES cells. As expected, up to 30% of the cultured cells were found to express HC-related markers, including Math1, myosin6, myosin7a, calretinin, α9AchR, and Brn3c (also known as Pou4f3), with remarkable formation of stereocilia-like structures.
    Materials and methods
    Results
    Discussion Although several reports have documented differentiation of ES cells into inner ear HCs in vitro, the methods employed in those studies were composed of multiple steps and the resulting induction efficiency of HCs was quite low (Oshima et al., 2010; Li et al., 2003). Recently, we reported a simple two-step method for more efficient induction of inner ear HC-like cells from mouse ES cells using ST2 stromal cell conditioned medium (CM), termed the HIST2 method, which was shown capable to generate cells immuno-positive for the HC-related markers Math1, myosin6, myosin7a, calretinin, α9AchR, and Brn3c in approximately 20% of the cultured cell populations (Ouji et al., 2012). In another study, we introduced the Math1 gene, a key transcription factor for HC differentiation and development, into ES cells under a tetracycline-controlled condition, which resulted in generation of HC-like cells among approximately 10% of cultured Math1-ES cells (Ouji et al., 2013). In the present study, we used the HIST2 method in combination with Math1-ES cells and found that approximately 30% of those cells were induced into an HC-like population. The combined method used in the present study resulted in more efficient induction of ES-derived HC cells, as compared to the previously reported 2D in vitro induction method (Oshima et al., 2010) and the methods used in our other studies (Ouji et al., 2012, 2013). HCs exist along with supporting cells (SCs) in the cochlea and vestibular systems of the inner ear (Kwan et al., 2009; Whitfield, 2015; Walters and Zuo, 2013). Recently, gene expression profiling of HCs and surrounding cells (considered to be SCs) during mouse inner ear development was investigated, and the authors reported that some genes were preferentially expressed in cochlea and vestibular HCs, while others were found expressed in cochlea and vestibular SCs (Scheffer et al., 2015). To elucidate whether the HC-like cells induced using the present combined method were cochlear or vestibular type, 5 genes were selected for investigation; Grp (marker of cochlear and vestibular HCs), Lmod3 (marker for cochlear HCs), Emcn (marker for vestibular HCs), Dnah5 (marker for cochlear SCs), and Ptgds (for vestibular SCs). Use of the HIST2 method led to increased expressions of all 5 genes (Fig. 3), suggesting induction of both cochlear- and vestibular-type cells. In contrast, Math1 induction by Dox without the HIST2 method induced mRNA expression of the cochlear markers Lmod3 and Emcn, but not of the vestibular markers Dnah5 and Ptgds, suggesting that continuously forced expression of the Math1 gene, as applied in the present study, might be suitable to preferentially obtain cochlear-type cells including HC-like cells from ES cells. Math1 expression is an indicator of the pro-sensory region during development of the inner ear and necessary for HC differentiation (Chen et al., 2002; Jahan et al., 2015; Woods et al., 2004; Izumikawa et al., 2005). Therefore, utilizing the Tet-On system we added Math1 gene induction to the HIST2 method as an attempt to more efficiently obtain inner ear HCs from ES cells. As expected, the largest number of cells immunopositive for the examined HC markers was seen with the combined method (Fig. 1). In addition, both cochlear- and vestibular-HC marker genes were expressed at the highest level with our combined method (Fig. 3).