Controlling differentiation and proliferation in human stem cells intended for therapeutic use

Our ability to hear depends on auditory sensory hair cells and their associated neurons that reside in the cochlea. The exquisite sensitivity of the inner ear comes with the risk for damage for example by noise trauma, ototoxic drug damage, infections, age-related degeneration and genetic causes. Once lost, the sensory hair cells are never replaced resulting in chronic hearing impairment. Hearing impairment is a devastating and highly prevalent disorder with widespread implications for the individual and society as a whole. Adult onset hearing loss alone ranks among the five leading causes of burden of disease in Europe entailing enormous socio-economic costs. Prosthetic treatment with hearing aids and cochlear implants is limited in restoring normal hearing, and reaches only every fifth patient. Hearing aid amplification or cochlear implant stimulation often fail to improve language comprehension and hence perform unsatisfactory.

OTOSTEM addressed this urgent and unmet medical need for causal hearing loss therapies by focusing on human stem cell technology.

OTOSTEM developed two strategies using human stem cells for curing hearing loss: 1) stem cell-derived cellular agents for cell-based therapy and 2) stem cell-derived assays as a screening tool to identify novel drugs. To be safe and effective for therapy, transplantable human otic stem cells must be reliably produced in both purity and quantity. Novel procedures for such high pure production need to be developed. Likewise, identification of drug candidates requires extensive in vitro and in vivo investigations. OTOSTEM intends to bring new therapies to the point of entering a pre-clinical developmental stage in order to prepare grounds for clinical development.

Workpackage 5

In vivo investigations of stem cell and drug based therapies in hearing loss models

In WP5, in vivo hearing loss models were used to investigate efficacy of cell- and drug-based therapies. The four different rodent species employed are ideal in vivo models for a wide range of human hearing conditions. At the etiological level the main causes of hearing loss including infection, drug induced ototoxicity, noise induced trauma, and age related degeneration are addressed. At the cellular level the loss of sensory hair cells as well as auditory neurons is targeted.

Workpackage 4

Drug screening aiming at otoprotection and otoregeneration

Human otoprotection and otoregeneration bioassays do not exist. This technology is unprecedented in the field of translational hearing research and provides a solid platform for screening efforts and for further validation in vitro and in vivo (WP5). In WP4, we worked on bioassays capable of identifying (a) otoprotective compounds that will prevent death of human hair cells, and otoregenerative compounds that stimulate hair cell regeneration.

Workpackage 3

Hearing loss in a dish model and ototoxicity

Human ototoxicity bioassays do not exist. There is, however, an obvious need for ototoxicity screening. Recent zebrafish-based screening efforts demonstrated that 21 FDA-approved drugs out of 1,040 well- known bioactive compounds (NINDS Custom Collection II) display selective toxicity to lateral line sensory hair cells. It is presumed that many drugs that are currently in use or in various stages of clinical trials have ototoxic potential. Ototoxicity tests with human hESCs and hiPSCs derived inner ear cells were established with aminoglycosides and cisplatin – known ototoxic drugs that cause hearing loss and are in widespread clinical use.

Workpackage 2

Otic progenitors cells suitable for cell transplantation

For cell transplantation purposes, enriched otic progenitors were differentiated along sensory vs. neural lineages depending on the targeted tissue for cell replacement and tested for their tumorigenic potential. Teratoma formation capacity of enriched otic progenitors versus the non-enriched heterogeneous population was investigated by injection into severe combined immunodeficient (SCID) mice.
The endpoint of WP2 was to provide enriched and proven non-tumorigenic otic progenitor cells with the capacity to potentially restore the hearing function by cell transplantation in in vivo animal models (WP5).

Workpackage 1

Controlling differentiation and proliferation in human otic stem cells

The main objective of WP1 was to provide a solid foundation for the whole project by defining the core cell type of the assay, the purified otic progenitor cell. This was based on functional and differentiation abilities, at the single cell transcriptional level and other important cell biological parameters such as surface markers and proliferative potential. The targeted result was to obtain reliable cell-based assays using bankable progenitor cells that are generated with highly reproducible techniques.

OTOSTEM has identified the principal technological hurdles for the development of cell-based and drug-based hearing loss therapies and addressed these hurdles in successive and systematic manner. Otic progenitor populations were differentiated into cochlear cell types such as sensory cells, supporting cells and neurons. The cross validation from different stem cell sources (hESCs, hiPSCs, native human fetal and adult inner ear stem cells) represented a unique opportunity and particular strength of the OTOSTEM consortium that will provide an unprecedented potential for optimization and selection of the most suitable human stem cell protocols to generate otic progenitors and differentiated cells.