Statement of Benefit to California:
Year 1The main goals of our research are to establish an experimental protocol for coaxing embryonic stem cells into inner ear sensory cells (sensory hair cells). This work has implications on future treatment of hearing loss and balance disorders, for which no current treatment exist. We began with using mouse embryonic stem cells to explore specific experimental conditions that lead to cell differentiation in direction of the inner ear. In the last funding period, we were able to increase the efficacy of this procedure by approximately 10-fold. At the same time, we were able to cut the time needed for a guidance experiment from 2-3 months to 8 days. We have now begun testing whether human embryonic stem cells are also able to follow the same guidance protocol and we found that human cells can also coaxed toward the inner ear lineage with a slightly modified protocol. The efficiency with human cells is comparable to the efficiency experienced with the mouse cells. The time needed for initial differentiation of the human cells into early inner ear is not very much different from the time needed for differentiating mouse cells, but we still need to verify this preliminary result.
In addition to in vitro guidance tests, we have begun with assessing the in vivo potential of mouse and human inner ear precursor cells, which were derived from mouse and human embryonic stem cells. Toward this goal, we have begun to establish a coculture system in which we culture the mouse and human cells inside the developing inner ear of chicken embryos. Before starting with the more complicated in ovo experiments, we decided to first establish a culture system in vitro, which will allow us to perform more pilot tests in a more controlled cell culture environment. Establishment and initial assays in this regard have been conducted in the past funding period and we are optimistic that we will be able to assess the potential of mouse and human embryonic stem cell-derived inner ear cell types using the established in vitro tests in the upcoming funding period.
Year 2In the last year, we have achieved a major milestone by showing that it is possible to generate functional inner ear sensory cells (hair cells) from mouse embryonic stem cells using a guidance protocol that was completely conducted in vitro (in cell culture). The goal of this project is to achieve the same result with human cells at the end of the funding period. We are on track with this endeavor, but we have encountered a number of quite substantial roadblocks. The major roadblock is that human cells appear to require much more time to differentiate into mature inner ear cell types when compared with mouse cells. We are currently working on a protocol that allows us to provide a sustained stimulation of the cell signaling pathways that are needed to keep human cells on track to develop along the otic (inner ear) pathway. We have learned a lot about how human embryonic stem cells react when exposed to various signaling environments and we have made the discovery that the embryonic stem cells are able to differentiate relatively quickly into early embryonic cells (lineages), but that the development of later cell types (i.e. organogenesis), does require quite some time.
Year 3The overarching goal of this grant was to develop an experimental protocol for coaxing human embryonic stem cells to develop into inner ear sensory hair cells. This work has implications on future treatment of hearing loss and balance disorders, for which no current treatment exist. Generation of sensory hair cells and their accompanying so-called supporting cells is a complex endeavor because these cells develop from an embryonic structure called the otic placode. Generation of placodal cells is quite complex and appears not to be a default pathway, which can be explored for generation of myocytes or neural cells. Another aggravating factor was that human cells appear to require a different guidance protocol than mouse ESCs.
With 4 years of CIRM funding, we were able to overcome most of the obstacles that we encountered along the way and we are now at a point where we are able to generate human hair cell-like cells in the culture dish. These cells express appropriate marker proteins and they display the cytomorphological specializations that you would expect to find in a sensory hair cell. Still, more experiments are needed to ensure that the cells function properly. These analyses are complicated and they require an additional manipulation of human ESCs (adding a transgenic reporter). This work is still ongoing and it is required for publishing the results of the study.