Enhanced survival of hypoimmunogenic otic progenitors following intracochlear xenotransplantation: repercussions for stem cell therapy in hearing loss models.

Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments.

Bridging the electrode-neuron gap: finite element modeling of in vitro neurotrophin gradients to optimize neuroelectronic interfaces in the inner ear.

Although cochlear implant (CI) technology has allowed for the partial restoration of hearing over the last few decades, persistent challenges (e.g., poor performance in noisy environments and limited ability to decode intonation and music) remain. The "electrode-neuron gap" is inherent to these challenges and poses the most significant obstacle to advancing past the current plateau in CI performance. We propose the development of a "neuro-regenerative nexus"-a biological interface that doubly preserves native spiral ganglion neurons (SGNs) while precisely directing the growth of neurites arising from transplanted human pluripotent stem cell (hPSC)-derived otic neuronal progenitors (ONPs) toward the native SGN population. We hypothesized that the Polyhedrin Delivery System (PODS®-recombinant human brain-derived neurotrophic factor [rhBDNF]) could stably provide the adequate BDNF concentration gradient to hPSC-derived late-stage ONPs to facilitate otic neuronal differentiation and directional neurite outgrowth. To test this hypothesis, a finite element model (FEM) was constructed to simulate BDNF concentration profiles generated by PODS®-rhBDNF based on initial concentration and culture device geometry. For biological validation of the FEM, cell culture experiments assessing survival, differentiation, neurite growth direction, and synaptic connections were conducted using a multi-chamber microfluidic device. We were able to successfully generate the optimal BDNF concentration gradient to enable survival, neuronal differentiation toward SGNs, directed neurite extension of hPSC-derived SGNs, and synaptogenesis between two hPSC-derived SGN populations. This proof-of-concept study provides a step toward the next generation of CI technology. STATEMENT OF SIGNIFICANCE: Our study demonstrates that the generation of in vitro neurotrophin concentration gradients facilitates survival, neuronal differentiation toward auditory neurons, and directed neurite extension of human pluripotent stem cell-derived auditory neurons. These findings are indispensable to designing a bioactive cochlear implant, in which stem cell-derived neurons are integrated into a cochlear implant electrode strip, as the strategy will confer directional neurite growth from the transplanted cells in the inner ear. This study is the first to present the concept of a "neuro-regenerative nexus" congruent with a bioactive cochlear implant to eliminate the electrode-neuron gap-the most significant barrier to next-generation cochlear implant technology.

Three-Dimensional Otic Neuronal Progenitor Spheroids Derived from Human Embryonic Stem Cells.

Stem cell-replacement therapies have been proposed as a potential tool to treat sensorineural hearing loss by aiding the regeneration of spiral ganglion neurons (SGNs) in the inner ear. However, transplantation procedures have yet to be explored thoroughly to ensure proper cell differentiation and optimal transplant procedures. We hypothesized that the aggregation of human embryonic stem cell (hESC)-derived otic neuronal progenitor (ONP) cells into a multicellular form would improve their function and their survival in vivo post-transplantation. We generated hESC-derived ONP spheroids-an aggregate form conducive to differentiation, transplantation, and prolonged cell survival-to optimize conditions for their transplantation. Our findings indicate that these cell spheroids maintain the molecular and functional characteristics similar to those of ONP cells, which are upstream in the SGN lineage. Moreover, our phenotypical, electrophysiological, and mechanical data suggest an optimal spheroid transplantation point after 7 days of in vitro three-dimensional (3D) culture. We have also developed a feasible transplantation protocol for these spheroids using a micropipette aided by a digital microinjection system. In summary, the present work demonstrates that the transplantation of ONP cells in spheroid form into the inner ear through micropipette 7 days after seeding for 3D spheroid culture is an expedient and viable method for stem cell replacement therapies in the inner ear.

An engineered three-dimensional stem cell niche in the inner ear by applying a nanofibrillar cellulose hydrogel with a sustained-release neurotrophic factor delivery system.

Although the application of human embryonic stem cells (hESCs) in stem cell-replacement therapy remains promising, its potential is hindered by a low cell survival rate in post-transplantation within the inner ear. Here, we aim to enhance the in vitro and in vivo survival rate and neuronal differentiation of otic neuronal progenitors (ONPs) by generating an artificial stem cell niche consisting of three-dimensional (3D) hESC-derived ONP spheroids with a nanofibrillar cellulose hydrogel and a sustained-release brain-derivative neurotrophic factor delivery system. Our results demonstrated that the transplanted hESC-derived ONP spheroids survived and neuronally differentiated into otic neuronal lineages in vitro and in vivo and also extended neurites toward the bony wall of the cochlea 90 days after the transplantation without the use of immunosuppressant medication. Our data in vitro and in vivo presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear. Using our protocol to create an artificial stem cell niche in the inner ear, it is now possible to work on integrating transplanted hESC-derived ONPs further and also to work toward achieving functional auditory neurons generated from hESCs. Our findings suggest that the provision of an artificial stem cell niche can be a future approach to stem cell-replacement therapy for inner-ear regeneration. STATEMENT OF SIGNIFICANCE: Inner ear regeneration utilizing human embryonic stem cell-derived otic neuronal progenitors (hESC-derived ONPs) has remarkable potential for treating sensorineural hearing loss. However, the local environment of the inner ear requires a suitable stem cell niche to allow hESC-derived ONP engraftment as well as neuronal differentiation. To overcome this obstacle, we utilized three-dimensional spheroid formation (direct contact), nanofibrillar cellulose hydrogel (extracellular matrix), and a neurotrophic factor delivery system to artificially create a stem cell niche in vitro and in vivo. Our in vitro and in vivo data presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear.