Establishment Of Nerve Cell Model Derived From Connexin 26 Deficiency Deafness Patients And Mechanism Study

Defeciency of connexins could cause dysfunction of gap junction.In all connexin-associated diseases,deafness is one of the most important diseases for the high frequency in human population.Connexin 26(Cx26),which is encoded by gene Gap Junction Protein beta 2(GJB2),have been shown to account for a large proportion of congenital deaf cases in every population tested,linking with either nonsyndromic or syndromic sensorineural hearing loss.The 235delC mutation of Cx26 is the most frequent mutation in East Asian groups,with a carrier frequency of approximately 1%.Research on the pathogenesis of Cx26 deficiency deafness is hampered by the inaccessibility of the human cochlea and acoustic nerve.So the underlying mechanisms for Cx26 deficiency deafness still remain unclear.In mouse,extensive deletion may even be embryo lethal because of the decreased transplacental glucose uptake.In human,deficiency of Cx26 won't influence transplacental glucose uptake,because of the different human placental barrier.Cx26 deficiency won't be lethal in human embryo stage in most cases.In the long time of human hearing development,it should be illuminated that,when and in which kind of cell Cx26 deficiency would firstly play a great influence.Recently,induced pluripotent stem cell(iPSC)technique has matured to be possible to address the inaccessibility of the human disorders.The methods for non-integration reprogramming to get iPSCs by transfecting modified plasmids,mRNA,miRNA and episomes overcome the inherent problems incurred by introducing viral vectors to target cells.The non-integration reprogramming system also eliminated the biosafety risks surrounding viral-based systems.In this paper,we focused on studys as below:1.Fibroblasts were taken from profound deaf patients caused by Cx26 deficiency,and were induced to non-integration induced pluripotent stem cell lines.Five hESC-like colonies were picked individually and disaggregated into small clumps without enzymatic digestion for passage.After several passages,three iPSC lines(named iPSC-1,iPSC-2,iPSC-3),were established respectively.,whose morphology,internal and external gene expression were characterized.2.To evaluate the differentiation capacity of Cx26 deficiency iPSCs in vitro,we allowed iPSC-1 and iPSC-2 to differentiate into embryoid bodies(EBs)for 7 days(Fig.S4A).The RT-PCR results confirmed that these differentiated cells expressed key marker genes of the three germ layers,such as microtubule-associated protein 2(MAP2,ectoderm),paired box 6(PAX6,ectoderm),Msh homeoboxl(MSX1,mesoderm)and SRY-box containing gene 17(SOX17,endoderm).To evaluate the differentiation capacity in vivo,1×106 cells of iPSC-1 and iPSC-2 were subcutaneously injected into severe combined immune deficiency(SCID)mice,respectively.Four weeks later,the teratomas were harvested for hematoxylin-eosin(HE)staining.Histological examination showed that the teratomas contained derivatives of the endoderm(glandular structures),mesoderm(cartilage),and ectoderm(epithelium,nerve fibers).These results indicated that Cx26 deficiency iPSC-1 and iPSC-2 are pluripotent stem cells.3.To investigate the effect of Cx26 deficiency in the process of hearing development,we investigated whether human iPSCs with GJB2 235delC mutation are able to differentiate into neural progenitors and neurons in vitro,followed a published protocol with small modification.The pluripotent stem cells were differentiated into suspended EBs when withdraw of feeder cells and bFGF,cultured with low-attached dishes.Day 7 EBs were replated and further differentiated to form neuroepithelial(NE)cells.Typical cellular structures "neural tube-like rosettes" were appeared 12-18 days later.In the process of differentiation,neuroepithelia cells appeared,which were characterized by columnar epithelial morphology and stained highly positive for neuroectodermal transcription factors PAX6 and NESTIN in D10.To purify the neuroepithelial cells,neural tube-like rosettes were separated and grown as neural spheres for several days.PAX6 and N-CADHERIN,highly expressed in neuroepithelial cells,were up-regulated after neural tube formation,while the level of pluripotent transcription factor NANOG dropped significantly in neural progenitor cells(NPCs)in D20.NEUN;??-tubulin(TUBB3)both expressed in NPCs.The neural spheres were replated for neuronal differentiation until the synapse-like structures were formed.In D42,a portion of pluripotent stem cells-derived neural cells grew like astrocytes and highly expressed GFAP.Some of the other neural cells in D42 showed typical synapse as neurons,and highly expressed neuronal differentiation marker TUBB3.We found that Cx26(GJB2)was expressed in certain kinds of human cells,including hESCs,EBs,neural cells and iPSCs,while the expression of Cx32(GJB1)was surprised highly up-regulated in iPSCs.No obvious difference in morphology was observed between hESC-derived neural cells and Cx26 deficiency iPSC-derived neural cells.These data indicated that Cx26 deficiency iPSCs derived from donor cells could differentiate into neural progenitor cells and neurons in vitro in the similar manner to hESCs,not only in morphology but also in endogenous gene expression and surface markers,except the Cx32(GJB1).