Modeling human early otic sensory cell development with induced pluripotent stem cells

PLOS ONE, Nov 2019

The inner ear represents a promising system to develop cell-based therapies from human induced pluripotent stem cells (hiPSCs). In the developing ear, Notch signaling plays multiple roles in otic region specification and for cell fate determination. Optimizing hiPSC induction for the generation of appropriate numbers of otic progenitors and derivatives, such as hair cells, may provide an unlimited supply of cells for research and cell-based therapy. In this study, we used monolayer cultures, otic-inducing agents, Notch modulation, and marker expression to track early and otic sensory lineages during hiPSC differentiation. Otic/placodal progenitors were derived from hiPSC cultures in medium supplemented with FGF3/FGF10 for 13 days. These progenitor cells were then treated for 7 days with retinoic acid (RA) and epidermal growth factor (EGF) or a Notch inhibitor. The differentiated cultures were analyzed in parallel by qPCR and immunocytochemistry. After the 13 day induction, hiPSC-derived cells displayed an upregulated expression of a panel of otic/placodal markers. Strikingly, a subset of these induced progenitor cells displayed key-otic sensory markers, the percentage of which was increased in cultures under Notch inhibition as compared to RA/EGF-treated cultures. Our results show that modulating Notch pathway during in vitro differentiation of hiPSC-derived otic/placodal progenitors is a valuable strategy to promote the expression of human otic sensory lineage genes.

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Modeling human early otic sensory cell development with induced pluripotent stem cells

June Modeling human early otic sensory cell development with induced pluripotent stem cells Hanae Lahlou 0 1 2 Alejandra Lopez-Juarez 0 1 2 Arnaud Fontbonne 0 1 2 Emmanuel Nivet 0 2 Azel Zine 0 1 2 0 Current address: Harvard Medical School, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary , Boston , United States of America 1 Aix Marseille Universit e , CNRS, LNIA UMR 7260, Marseille, France, 2 Aix Marseille Universit eÂ, CNRS, NICN UMR 7259, Marseille , France , 3 Universite de Montpellier, Facult e de Pharmacie , Montpellier , France 2 Editor: Bruce B Riley, Texas A&M University , UNITED STATES The inner ear represents a promising system to develop cell-based therapies from human induced pluripotent stem cells (hiPSCs). In the developing ear, Notch signaling plays multiple roles in otic region specification and for cell fate determination. Optimizing hiPSC induction for the generation of appropriate numbers of otic progenitors and derivatives, such as hair cells, may provide an unlimited supply of cells for research and cell-based therapy. In this study, we used monolayer cultures, otic-inducing agents, Notch modulation, and marker expression to track early and otic sensory lineages during hiPSC differentiation. Otic/placodal progenitors were derived from hiPSC cultures in medium supplemented with FGF3/ FGF10 for 13 days. These progenitor cells were then treated for 7 days with retinoic acid (RA) and epidermal growth factor (EGF) or a Notch inhibitor. The differentiated cultures were analyzed in parallel by qPCR and immunocytochemistry. After the 13 day induction, hiPSC-derived cells displayed an upregulated expression of a panel of otic/placodal markers. Strikingly, a subset of these induced progenitor cells displayed key-otic sensory markers, the percentage of which was increased in cultures under Notch inhibition as compared to RA/EGF-treated cultures. Our results show that modulating Notch pathway during in vitro differentiation of hiPSC-derived otic/placodal progenitors is a valuable strategy to promote the expression of human otic sensory lineage genes. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The research leading to these results has received funding from the European Community's Seventh Framework Programme under grant agreement No. 603029 (Project OTOSTEM). ªLa Fondation Pour l'Auditionº provided support for the final year Ph.D. fellowship rewarded to HL." The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Hearing loss and vestibular dysfunction are the most common sensory deficits in humans [ 1 ]. The inner ear is a highly specialized sensory organ containing auditory and vestibular hair cells (HCs) that transduce mechanical energy into electrical energy for transmission to the central nervous system [ 2 ]. During otic development, HCs in the inner ear are derived from the differentiation of early otic progenitor cells through a precise temporally and spatially-coordinated pattern of gene expression orchestrated by complex signaling cascades [3_4]. A normal human cochlea contains approximately 16,000 sensory HCs forming one row of inner HCs and three rows of outer HCs. They are limited in number and are susceptible to damage from a variety of insults, ranging from ototoxic drugs to loud noise exposure, genetic mutations, or the effects of aging. In contrast to the avian cochlea able to regenerate lost HCs [5±6], the mature mammalian cochlea is unable to spontaneously regenerate HCs leading to permanent hearing loss. Over the past few years, stem cell-based therapy approaches aiming to emulate otic development in the production of HCs from stem cells have received substantial interest [7±8]. The generation of replacement HCs from a renewable source of otic progenitors remains one of the principal requirements for the successful development of a cell-based therapy within the inner ear. Murine embryonic stem cells (mESCs) have already demonstrated their capability of differentiating into otic epithelial lineage in vitro [9±15]. Furthermore, previous studies with human embryonic stem cells (hESCs) have revealed their ability to differentiate along an otic neurogenic lineage, giving rise to neurons with a partial functional restoration of HC innervation in an animal model of auditory neuropathy [16±17]. There is also evidence that hESCs are able to differentiate into cells of otic epithelial lineage when grown in aggregate/embryoid body (EB)- or adherent cell cultures [18±19]. Recently, the concept of differentiating hESC-derived HC-like cells has been elegantly demonstrated by the ability of these hESCs to differentiate self-guided when cultured in hydrogels as extracellular matrix mimics for three-dimensional (3D) cell culture [ 20 ]. These EB/aggregate and 3D-organoid in vitro guidance methods did allow (...truncated)


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Hanae Lahlou, Alejandra Lopez-Juarez, Arnaud Fontbonne, Emmanuel Nivet, Azel Zine. Modeling human early otic sensory cell development with induced pluripotent stem cells, PLOS ONE, 2018, Volume 13, Issue 6, DOI: 10.1371/journal.pone.0198954