Objectives: Hair cells and neurons of the inner ear are a specialized group of neurosensory population critical for hearing and balance. Specification of this group of cells includes the upregulation of key otic markers such as the transcription factor SOX2. In mammals, however, the regenerative capacity of these cells is limited compared to birds and amphibians.
Methods: To unravel the underlying transcriptional profile, a cell isolation protocol was optimized by combining an indirect immunofluorescence approach with laser microdissection. Cryosections were obtained from human fetuses ranging from 6 to 13 weeks gestation. Selected tissue sections at specific intervals were firstly labelled with SOX2 and GATA3 for identification of the pro-sensory domain and the innervating neurons of the cochlea and vestibular apparatus. By extrapolating the expression pattern of the selected markers, laser microdissection was then performed on corresponding tissue sections which were stained with cresyl violet.
Results: Analysis of RNA extracted from the collected cell population revealed that RNA integrity was intact and of sufficient yield. Additionally, upregulation of key hair cell markers such as ATOH1 and POU4F3 at the cochlear sensory epithelium at 12 weeks gestation, and NEUROD1 and SHH in 7 to 8 weeks spiral ganglion neurons provided a validation for the reliability of the cell isolation technique.
Conclusion: Overall, the protocol permits the isolation of target cell population within its native landscape whilst preserving the RNA integrity for further downstream analysis.
Keywords: inner ear; laser microdissection; otic progenitors; prosensory domain
Ethical statement: Human fetal samples were collected from medically terminated pregnancies. This was performed following the full approval of the Health Research Authority and Research Ethics Committee (HRA/REC) (IRAS application ID 193822, Sheffield number STH1908) and in adherence to the Polkinghorne guidelines/Human Tissue Act (HTA). Informed consent was obtained from patients after the decision to terminate the pregnancy.
Funding statement: The study was funded by the scholarship from Ministry of Higher Education, Malaysia.