| Abstract |
Background: Familial Meniere's disease (FMD) is an inner ear disorder
defined by sensorineural hearing loss, episodic vertigo and tinnitus and
it is observed in 5-15% of MD cases. By whole-exome sequencing, we
identified two heterozygous single-nucleotide variants in FAM136A and DTNA
genes in a Spanish family with three affected cases in consecutive
generations, highly suggestive of autosomal-dominant inheritance. We have
generated an induced pluripotent stem cell line (hPSC; GENYOi007-A) from a
FMD patient with both mutations and differentiated them into 3D inner ear
organoids (IEO).
Methods: CytoTune 2.0 Reprogramming kit was used to reprogram peripheral
blood mononuclear cells from this FMD patient. Characterization of the
cell line GENYOi007-A included genetic analysis of DTNA and FAM136A
variants, Short Tandem Repeats profiling (STR), expression of pluripotency-
associated factors and differentiation studies in vitro. To begin the
differentiation to IEO, hPSC were aggregated and treated with
extracellular matrix proteins to promote epithelialization. Then, by
recapitulating signaling pathway activation and attenuation during inner
ear development we modulated signaling pathways inducing sequential
formation and subsequent self-guided morphogenesis to form sensory
epithelia containing hair cells and supporting cells, as well as neurons
forming synapses with the hair cells.
Results: First, we confirmed the presence of DTNA and FAM136A variants by
Sanger sequencing. GENYOi007-A silenced the expression of exogenous
transgenes and activated the expression of the endogenous pluripotent
transcription factors (SOX2, REX1, NANOG and OCT4). Importantly, GENYOi07-
A cells showed normal karyotype (46, XX). Furthermore, the expression of
the pluripotent markers SSEA4, Tra1-60 and Tra1-81 was confirmed by flow
cytometry analysis and Confocal imaging. Finally, to demonstrate its
capacity to differentiate into the three germ layers we performed an
embryoid bodies (EBs) formation assay. EBs derived from this cell line
showed specific expression of representative markers of the three germ
layers: ectoderm (beta3-tubulin), mesoderm (Vimentin) and endoderm
(Cytokeratin CKAE1-AE). IEO showed high levels of MYO7a (FC= 4.3); ATOH1
(FC=54.1) and TUBB3 (FC=13.4) when compared to the hPSC, demonstrating
their capacity to differentiate into inner ear tissue. Likewise, DTNA and
FAM136A had higher expression in the IEO (FC= 15.1 and 1.6, respectively).
Western blot supported the above results.
Conclusion: Both DTNA and FAM136A are expressed in inner ear tissue-like
organoids. Further experiments are needed to define the cell types
involved in FAM136A and DTNA expression in human inner ear tissue.
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