Purchase this article with an account.
JAEHO JUNG, Hyeshin Jeon, JEONGMIN KWON; Thyroid stimulating hormone (TSH) receptor expression in human extraocular muscle cells in in vitro primary culture. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2171.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In this study, we have achieved primary culture and myogenic differentiation of human EOM myoblasts (HEOMMs) in vitro, providing a novel tool for the study of EOM changes in disease, especially in TAO. We also detected the presence of the TSH receptor on HEOMMs and assayed TSH receptor expression level changes during myogenic differentiation.
HEOMMs were isolated from muscle samples by collagenase digestion. For each sample was minced and placed in collagenase solution and incubated at 37°C for 1 hour. Digested muscle solution was filteredand with complete Ham’s F10 medium with fetal bovine serum, antibiotics and basic fibroblast growth factor. After centrifuge, the pellet was seeded on a gelatin-coated dish. At confluence, HEOMMs were allowed to differentiate into myotubes on normal dishes with differentiation medium. Myogenic differentiation of HEOMMs was observed; myoblasts fused to form multi-nucleated myotubes.To verify the isolation of HEOMMs, we immunostained the cells for PAX7 and MYOD1. In addition, we investigated the expression of Shox2, a signature genetic marker of extraocular myoblasts, and Hoxc10, an exclusive marker of hind-limb muscle-derived myoblasts by PCR.To determine the presence of TSH receptor on the surface of HOEMs, we performed TSH receptor immunostaining and to investigate the TSH receptor protein level using western blot analysis.
HEOMMs were positive for PAX7 and MYOD1 staining, both markers of human myoblasts. The EOM-specific gene Shox2 was observed by PCR; however, the hind limb muscle-derived myoblast marker was not detected. Myotube formation began four to six days after induction of differentiation. The overall fusion index and myotube area, measures of myogenic activity, were highest seven days after induction of myogenic differentiation. Western blot analysis performed seven days after myogenic differentiation and HEOMMs were positive for the TSH receptor.
In this study, we have demonstrated human extraocular muscle myoblast isolation, culture, and differentiation to myotubes in vitro, and revealed that these cells express thyroid stimulating hormone (TSH) receptors. The presence of the TSH receptor, an autoimmune target, in extraocular muscle myoblasts suggests a possible target autoantigen for thyroid-associated ophthalmopathy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only