Survival of Partially Differentiated Mouse Embryon

Authors: Michael S Hildebrand HansHenrik M Dahl Jennifer Hardman Bryony Coleman Robert K Shepherd Michelle G de Silva

Publish Date: 2005/10/06

Volume: 6, Issue: 4, Pages: 341-354

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Abstract

The low regenerative capacity of the hair cells of the mammalian inner ear is a major obstacle for functional recovery following sensorineural hearing loss A potential treatment is to replace damaged tissue by transplantation of stem cells To test this approach undifferentiated and partially differentiated mouse embryonic stem ES cells were delivered into the scala media of the deafened guinea pig cochlea Transplanted cells survived in the scala media for a postoperative period of at least nine weeks evidenced by histochemical and direct fluorescent detection of enhanced green fluorescent protein EGFP Transplanted cells were discovered near the spiral ligament and stria vascularis in the endolymph fluid of the scala media In some cases cells were observed close to the damaged organ of Corti structure There was no evidence of significant immunological rejection of the implanted ES cells despite the absence of immunosuppression Our surgical approach allowed efficient delivery of ES cells to the scala media while preserving the delicate structures of the cochlea This is the first report of the survival of partially differentiated ES cells in the scala media of the mammalian cochlea and it provides support for the potential of cellbased therapies for sensorineural hearing impairmentTo achieve restoration of auditory function following sensorineural hearing loss it will be necessary to regenerate or replace sensory hair cells This is a complex task as the organ of Corti where the hair cells are located is difficult to access The scala media is protected by a robust blood–endolymph barrier that maintains the stability of the endolymph bathing the apical surface of the organ of Corti Juhn 1988 We therefore delivered embryonic stem ES cells directly into the scala media through the basilar membrane Using this approach we have achieved efficient delivery of ES cells into the scala media and have demonstrated their survival for at least nine weeks Importantly the structures of this highly regulated scalae were not adversely affected by the implantation of ES cells via this routeRecent advances in ES cell technology have allowed us to culture and differentiate stem cells in vitro Rathjen et al 1999 Lake et al 2000 Rathjen et al 2002 It is possible that these cells could be used to facilitate in vivo cell therapy of the inner ear potentially replacing sensory hair cells lost or damaged by loud sound exposure to ototoxic drugs Palomar Garcia et al 2001 aging or hereditary gene defects Kelsell et al 1997 Tekin et al 2001 Loss of mammalian hair cells is permanent and causes irreversible hearing defects in humans Palomar Garcia et al 2001 The discovery that hair cells of the avian basilar papilla BP the functional equivalent of the mammalian organ of Corti are regenerated after preexisting hair cells have been destroyed Corwin and Cotanche 1988 Ryals and Rubel 1988 Warchol and Corwin 1996 has stimulated much interest in the possibility of hair cell regeneration therapy in mammals Staecker and Van De Water 1998 The continuous formation of cochlear sensory epithelial cells with and without insult to the auditory system has also been demonstrated in the lower vertebrate inner ear but not in mammals Cotanche and Lee 1994 Stone and Rubel 2000 Reng et al 2001The delivery of ES cells to damaged tissues in vivo has been reported Björklund et al 2002 Rideout et al 2002 The objective of cell therapy in the inner ear is to restore auditory function by regenerating or replacing damaged or lost sensory hair cells auditory neurons and supporting cells One of the first reports of stem cell delivery to the inner ear was the study by Ito et al 2001 that demonstrated survival and migration of adult rat neural stem cells implanted into the scala tympani of the rat cochlea The betagalactosidase βGalexpressing cells migrated to the organ of Corti and some cells were shown to adopt hair celllike morphology and to stain with phalloidin that binds to the Factin in stereocilia and other structures Ito et al 2001 The authors speculated that if the stem cells could localize to the correct region of the cochlea then they would take on hair cell characteristics Ito et al 2001 However the correct targeting of stem cells to the organ of Corti alone is unlikely to be sufficient to promote hair cell development and differentiation as the appropriate developmental cues may not be present in the adult cochlea The partial differentiation of ES cells in vitro prior to implantation may provide these cells with the developmental potential to form new hair cellsSince then other groups have reported on the transplantation of ES cells into the inner ear Hu et al 2004 Sakamoto et al 2004 Sakamoto et al 2004 reported the survival of ES cells predominantly in the vestibular region of the mouse inner ear and also some cells in the scala media of the cochlear duct after transplantation for four weeks In comparison the study by Hu et al 2004 demonstrated the survival and migration of mouse ES cells along the auditory nerve after xenotransplantation into auditory nerve fibers ANFs of the rat cochlea They showed that the ES cells could survive for up to nine weeks and that they migrated along the ANFs into the brainstem Although these studies have demonstrated the survival of ES cells in the cochlea the efficiency with which cells are delivered or migrate to the scala media has been low and neither have examined the survival of partially differentiated cell typesThe formation of new hair cells may require predifferentiation of progenitor ES cells prior to their transplantation in vivo as the damaged cochlear sensory epithelium may not be able to provide all the necessary developmental signals Cochlear hair cells arise from neuroectodermal precursors and their development is dependent on regulation of many genes and growth factors Malgrange et al 2002 Pirvola et al 2002 ES cells cultured in the presence of the medium conditioned by the human hepatocarcinoma cell line HepG2 MEDII medium have been shown to form morphologically distinct primitive ectodermlike cellular aggregates EBMs Rathjen et al 1999 Rathjen et al 2002 Treatment of the aggregates with basic fibroblast growth factor bFGF directs the cells down the neuroectodermal lineage Torres and Giraldez 1998 In the current study a differentiation strategy was established in vitro to generate cells along the neuroectoderm pathway Cell types from this developmental model were selected for in vivo transplantation into the guinea pig cochlea to attempt cellular replacement or regeneration of auditory hair cells The cell types included undifferentiated ES cells ES cells partially differentiated in MEDII medium for three or seven days and ES cells partially differentiated over nine days with MEDII medium and bFGF The cells were delivered into 14 guinea pigs deafened by administration of aminoglycosides prior to implantation and into one normal hearing animal It was demonstrated that mouse ES cells could survive for at least nine weeks in the guinea pig cochlea and that they could localize in the scala media Some transplanted cells localized close to the organ of Corti There was no evidence of significant immunological rejection despite the absence of immunosuppression and the surgical approach for delivery of ES cells resulted in minimal extraneous trauma to the cochlea

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