Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Abstract

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal β-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

Activation of CHK1 in Supporting Cells Indirectly Promotes Hair Cell Survival

“…Differentiating iMOP cells were generated by withdrawing bFGF for 7 days, to promote cell cycle exit and differentiation (Jadali et al, 2016). To determine the proliferative capacity of proliferating or differentiating iMOP cultures, incorporation of the nucleotide analog EdU was used.…”

“…To identify signaling pathways that maintain hair cell survival, we used iMOP cells that can self-renew and differentiate into hair cells and supporting cells (Kwan et al, 2015 ). Differentiating iMOP cells were generated by withdrawing bFGF for 7 days, to promote cell cycle exit and differentiation (Jadali et al, 2016 ). To determine the proliferative capacity of proliferating or differentiating iMOP cultures, incorporation of the nucleotide analog EdU was used.…”

“…Immortalized multipotent otic progenitor (iMOP) cells were grown in suspension with DMEM/F12 (Life Technologies) containing B27 supplement (Life Technologies), 25 μg/ml carbenicillin and 20 ng/ml basic fibroblast growth factor (bFGF; Pepro Tech; Jadali et al, 2016 ). For differentiation experiments, cells were cultured for 3 or 7 days in the absence of bFGF depending on the experiment.…”

Single cell analysis of the inner ear sensory organs

“…, 2012 ) and the hematopoietic system ( Seita and Weissman, 2010 ), resulting in very detailed differentiation schemes and cellular lineage trees. The resulting genetic programs and fate maps of cell lineages have advanced the fields of in vitro-directed differentiation of embryonic stem cells (ESC) and Induced pluripotent stem cells (iPSC) to enable regenerative medicine ( Atala, 2015 ; Azadeh et al , 2016 ; Costa et al , 2015 ; Jessop et al , 2016 ; Sasai, 2013 ; Wobma and Vunjak-Novakovic, 2016 ; Wu and Hochedlinger, 2011 ).…”

Single-Cell Fluorescence Analysis of Pseudotemporal Ordered Cells Provides Protein Expression Dynamics for Neuronal Differentiation

“…iMOP cells were generated from SOX2 expressing cochlea progenitors and immortalized by transient expression of C-MYC (Kwan et al, 2015). Under appropriate culture conditions, iMOP cells differentiate into bipolar and pseudounipolar neurons (Jadali et al, 2016). Unlike other neurons that form complex axonal and dendritic branches, iMOP-derived neurons display bipolar or pseudounipolar neurites similar to SGNs in vivo (Kiang et al, 1982; Spoendlin and Schrott, 1989; Nayagam et al, 2011).…”