Controlling the exposure of mouse embryonic stem cells to signaling pathways and letting these cells differentiate in 3-D culture has allowed scientists to generate inner ear hair cells. The cells can be used to study inner ear development and genetic diseases that affect the ear, or to screen drugs.
Past attempts to derive inner ear cells from stem cells have mainly relied on flat culture dishes. But Eri Hashino, Karl Koehler, and their colleagues at Indiana University School of Medicine suspected that a 3-D culture method called “serum-free floating culture of embryoid body-like aggregates with quick reaggregation,” or SFEBq, might be just the trick to obtaining inner ear cells.
“This method was most recently used to develop mouse and human retinas,” said Koehler. “These are highly structured, well organized tissues, so we thought that the same culture method might be applicable to deriving the inner ear, which is also a very complex organ.”
The researchers knew that the sensory cells of the ear differentiate from the same group of embryonic tissues, cranial placodes, as the pain and touch sensing cells found in the head and neck. Past studies had shown that the signaling molecule bone morphogenetic protein (BMP) is vital in directing cells away from a brain or spinal cord fate to form cranial placodes. But the team knew that BMP had to be down-regulated after this step of differentiation to produce ear cells.
“We realized that in our culture method, we’d have to figure out the perfect time to first treat cells with BMP to steer them toward a non-neural fare, and then after that we’d have to inhibit BMP,” said Koehler.
Through trial and error, the team of researchers was able to find the perfect time to expose the culture to BMP and BMP inhibitor, as well as fibroblast growth factor (FGF) and transforming growth factor beta (TGF beta). Moreover, the scientists separately induced the stem cells in two different layers of the culture in an effort to mimic embryonic developmental layers.
“It’s a really quick, complex signaling mechanism and what we did is try to recreate that over a period of 24 to 48 hours,” Koehler said.
To determine whether the cells had properly differentiated, the researchers examined several molecules known to be expressed only in inner ear sensory cells. When the method worked, it gave higher yields of derived inner ear cells than previous techniques. The researchers cultured ~1500 cells, almost the number of cells found in a mouse’s ear. Most of the cells were vestibular—the subtype that senses changes in gravity—rather than cochlear cells that detect sound. But the team doesn’t yet know why one cell type predominates.
“It was really interesting to us that we only got vestibular cells,” said Koehler. “It might suggest that the vestibular state is a more default one.”
Koehler, Hasino, and their colleagues plan to follow up with studies on how the differentiation paths between vestibular and cochlear ear cells diverge, using their model to study more in depth the steps of inner ear development and what can go wrong during this process.
“We think that this model would be excellent for disease modeling,” said Koehler. “We can potentially induce mutations into the stem cells before we differentiate them and then figure out how to fix the problems we see.”
1. Koehler, K.R., Mikosz, A.M., Molosh, A.I., Patel, D., Hashino, E. (2013) Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature.