Promotion of In Vitro Hair Cell-like Cell Differentiation from Human Embryonic Stem Cells through the Regulation of Notch Signaling

Abstract

The Notch signaling pathway plays an important role in otic neurogenesis by regulating the differentiation of inner ear hair cells and supporting cells. Notch-regulated differentiation is required for the regeneration of hair cells in the inner ear. The temporal expression pattern of Notch ligands and receptors during in vitro hair cell-like cell differentiation from human embryonic stem cells (hESCs) was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vectors encoding short hairpin RNAs were used to silence JAG-1, JAG-2, and DLL-1, according to the temporal expression pattern of Notch ligands. Then, the effect of each ligand on the in vitro differentiation of hair cells was examined by RT-PCR, immunofluorescence, and scanning electron microscopy (SEM). The results showed that the individual deletion of JAG-2 or DLL-1 had no significant effect on the differentiation of hair cell-like cells. However, the simultaneous inhibition of both DLL-1 and JAG-2 increased the number of hair cell-like cells and decreased the number of supporting cells. JAG-2 and DLL-1 may have a synergistic role in in vitro hair cell differentiation.

The ndrg2 Gene Regulates Hair Cell Morphogenesis and Auditory Function during Zebrafish Development

“…Therefore, we speculated that the phenotype of HC loss emerging in the ndrg2 mutants was due to the defects of HC differentiation. Notch-mediated lateral inhibition is widely believed to regulate HC versus supporting cell specification while suppression of Notch signaling promotes the differentiation of HCs from the supporting cells [ 4 , 6 , 32 , 33 , 34 , 35 ]. To verify whether the ndrg2 gene affected HC differentiation through the Notch signaling pathway, a Notch γ-secretase inhibitor LY411575 was used to treat the ndrg2 mutants.…”

“…Development of HCs is an extremely complex and highly coordinated process and it is regulated by a series of key factors involving multiple signaling pathways, such as Notch/Wnt/Atoh1, MAPK, PI3K/Akt, calcium channels and oxidative stress/ROS [ 4 , 5 , 6 , 7 , 8 ]. The classical and conserved Notch signaling pathway plays critical roles in the development and regeneration of HCs [ 3 , 4 , 5 , 6 , 32 ]. Studies suggested that Notch-mediated lateral inhibition determined the cell fate of the sensory epithelium and modulated the HCs versus supporting cell specification by activation of Notch signaling to inhibit the neighboring HCs from differentiating into the same cell type, and then to adopt the alternative fate, that is, supporting cells [ 4 , 6 , 32 ].…”

“…The classical and conserved Notch signaling pathway plays critical roles in the development and regeneration of HCs [ 3 , 4 , 5 , 6 , 32 ]. Studies suggested that Notch-mediated lateral inhibition determined the cell fate of the sensory epithelium and modulated the HCs versus supporting cell specification by activation of Notch signaling to inhibit the neighboring HCs from differentiating into the same cell type, and then to adopt the alternative fate, that is, supporting cells [ 4 , 6 , 32 ]. It was known that HCs were regenerated via mitosis or direct transdifferentiation of the supporting cells [ 3 , 4 ].…”

Modeling, applications and challenges of inner ear organoid

“…The temporal expression patterns of Notch signals, a key signal in regulating hair cell differentiation, were detected and the possible coreregulated roles of Notch ligand JAG2 (jagged canonical Notch ligand 2) and DLL1 (delta-like canonical Notch ligand 1) were hypothesized to be anticipated in the differentiation of hair cells. 20 In another research, F I G U R E 3 Origins of inner ear organoids. Inner ear organoids can be induced from either pluripotent or mono-potent stem cells, including inner ear stem cells, ESCs, and iPSCs.…”

“…obtained differentiated inner ear progenitor cells cultured with N2, B27, FGF3 and FGF10 for 12 days using human ESCs cell line X1. The temporal expression patterns of Notch signals, a key signal in regulating hair cell differentiation, were detected and the possible co‐reregulated roles of Notch ligand JAG2 (jagged canonical Notch ligand 2) and DLL1 (delta‐like canonical Notch ligand 1) were hypothesized to be anticipated in the differentiation of hair cells 20 . In another research, human ESCs (WA25 cell line) were incubated in an E8 medium containing low concentrations of Matrigel and FGF to induce the differentiation of the ectoderm to form dense spherical cell clusters.…”