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.
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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)