title: Single-cell analysis delineates a trajectory toward the human early otic lineage.
abstract: Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Megan
authors.initials: M
authors.lastname: Ealy
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Ellwanger
authors.affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305.
authors.forename: Nina
authors.initials: N
authors.lastname: Kosaric
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Andres P
authors.initials: AP
authors.lastname: Stapper
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, Stanford University School of Medicine, Stanford, CA 94305; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305 hellers@stanford.edu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Heller
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D007758:Ear, Inner / Q000166:cytology* / Q000196:embryology; D004475:Ectoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D059010:Single-Cell Analysis / Q000379:methods*; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000235:genetics
chemical_list: D019485:Bone Morphogenetic Proteins; D005346:Fibroblast Growth Factors
keywords: ectoderm; gene expression analysis; inner ear; pluripotent stem cells; posterior placode