Researchers develop new approach for generating inner ear hair cells

Scientists have created a more efficient and controlled way to produce lab-grown inner ear hair cells than current methods allow, offering a new tool for hearing loss research.

The study, published today as a Reviewed Preprint in eLife, is described by the editors as important work, substantially advancing our understanding of the factors that can induce hair cell-like cells from human stem cells. They add that the evidence supporting the findings is compelling. 

Hearing loss affects hundreds of millions of people worldwide and often results from the loss of sensory hair cells in the inner ear – specialised cells that convert sound vibrations into electrical signals for the brain. These hair cells can be damaged by exposure to loud noise, certain medications or infections, and aging. In humans, once these hair cells die, they do not regenerate, meaning hearing loss is often irreversible. Research into how this could be countered has been limited by the inaccessibility of real human hair cells and the inefficiency of lab-based models. 

In earlier work, the authors showed that mouse cells can be reprogrammed into those that are more like hair cells using four transcription factors: Six1, Atoh1, Pou4f3, and Gfi1, collectively referred to as SAPG. However, this method relies on viral delivery, which poses challenges for consistency and scalability.

“In our previous mouse studies, we used retroviruses to deliver the four SAPG genes, which successfully reprogrammed the cells into hair cell-like cells,” explains lead author Robert Rainey, a postdoctoral researcher at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of University of Southern California, US. “However, viral delivery has major drawbacks. It’s hard to control the timing and level of gene expression, and it introduces variability that makes it difficult to scale up or adapt for consistent use across different systems. We sought to develop a more efficient approach that could work reliably in human cells.”

Rainey and colleagues engineered a stable human stem-cell line carrying a doxycycline-inducible version of the SAPG transcription factors. By adding the antibiotic doxycycline to the culture, this method allowed precise control of the reprogramming process. To track when the cells began to take on hair cell characteristics, they included a fluorescent reporter gene that switched on as reprogramming progressed. 

When doxycycline was added, the team observed the first signs of reprogramming within three days. By day seven, around 35–40% of the cells expressed key hair cell gene markers such as MYO7A, MYO6, and POU4F3. This represented a more than 19-fold increase in efficiency compared to their previous virus-based approach, in half the time. 

The team then used single-cell RNA sequencing to analyse the gene expression patterns of the reprogrammed cells. They found that the cells shared many key transcription factors and signalling pathways with human fetal inner ear hair cells. Although the cells did not clearly separate into cochlear (hearing-related) or vestibular (balance-related) subtypes, they expressed markers of both. These results suggest that their method had produced cells that closely resembled immature, or early-developmental stage human hair cells.

The researchers also tested the electrical properties of the reprogrammed cells to see how closely they mimicked real hair cells. Using patch-clamp recordings, they found a range of voltage-gated ion currents – electrical signals generated by the movement of charged particles across the cell membrane, which are essential for how hair cells detect and respond to sound – including some that matched those seen in developing human hair cells. 

“This method offers several advantages over viral delivery systems. We can precisely control when reprogramming starts and stops by adding short pulses of doxycycline, and it works in standard two-dimensional cell cultures,” says corresponding author Andrew Groves, at the time of the study a Professor in the Department of Neuroscience and of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, US, and now head of the Department of Developmental Biology at Washington University School of Medicine, Missouri, US. “Together with eliminating the need for viral delivery, these features make the method far more scalable and adaptable for future research – whether for modelling human inner ear development, studying hearing loss, or screening for protective drugs.”

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