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Survival of human embryonic stem cells implanted in the guinea pig auditory epithelium

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ABSTRACT

Hair cells in the mature cochlea cannot spontaneously regenerate. One potential approach for restoring hair cells is stem cell therapy. However, when cells are transplanted into scala media (SM) of the cochlea, they promptly die due to the high potassium concentration. We previously described a method for conditioning the SM to make it more hospitable to implanted cells and showed that HeLa cells could survive for up to a week using this method. Here, we evaluated the survival of human embryonic stem cells (hESC) constitutively expressing GFP (H9 Cre-LoxP) in deaf guinea pig cochleae that were pre-conditioned to reduce potassium levels. GFP-positive cells could be detected in the cochlea for at least 7 days after the injection. The cells appeared spherical or irregularly shaped, and some were aggregated. Flushing SM with sodium caprate prior to transplantation resulted in a lower proportion of stem cells expressing the pluripotency marker Oct3/4 and increased cell survival. The data demonstrate that conditioning procedures aimed at transiently reducing the concentration of potassium in the SM facilitate survival of hESCs for at least one week. During this time window, additional procedures can be applied to initiate the differentiation of the implanted hESCs into new hair cells.

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ABR thresholds and histologic changes after unilateral neomycin deafening.(a) There was statistically significant elevation of thresholds overall (MANOVA, p < 0.01) and at each tested frequency (p < 0.05). Epi-fluorescence of auditory epithelium stained by phalloidin (actin, red) showed diverse tissue morphologies: scar formation (b), transition from scar formation to flat epithelium (c) or flat epithelium (d), but always complete hair cell loss. Scale bars are 25 μm.
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f2: ABR thresholds and histologic changes after unilateral neomycin deafening.(a) There was statistically significant elevation of thresholds overall (MANOVA, p < 0.01) and at each tested frequency (p < 0.05). Epi-fluorescence of auditory epithelium stained by phalloidin (actin, red) showed diverse tissue morphologies: scar formation (b), transition from scar formation to flat epithelium (c) or flat epithelium (d), but always complete hair cell loss. Scale bars are 25 μm.

Mentions: Clinically, candidate ears for stem cell therapy will have profound deafness due to a complete loss of hair cells. Therefore, we used deafened guinea pigs with widespread hair cell loss and profound deafness. We confirmed the outcome of the deafening insult using ABRs, measured one week after the unilateral deafening surgery. Statistically elevated ABR thresholds were obtained after deafening with neomycin (MANOVA; F = 174.79, df = 3, p < 0.001). Deafened animals had elevated thresholds at all frequencies (Fig. 2a). Residual hearing function was observed and probably is due to transmission of the sound stimulus to the undamaged (right) ear25. In the left ears, hair cells were invariably eliminated by the procedure. Supporting cells responded with differing degrees of pathological changes. In some areas, supporting cells remained in their normal height and exhibited the typical phalangeal scar formation (Fig. 2b). In other areas, we observed a transitional stage between scar formation and a flat epithelium (Fig. 2c), or flat epithelium (Fig. 2d).


Survival of human embryonic stem cells implanted in the guinea pig auditory epithelium
ABR thresholds and histologic changes after unilateral neomycin deafening.(a) There was statistically significant elevation of thresholds overall (MANOVA, p < 0.01) and at each tested frequency (p < 0.05). Epi-fluorescence of auditory epithelium stained by phalloidin (actin, red) showed diverse tissue morphologies: scar formation (b), transition from scar formation to flat epithelium (c) or flat epithelium (d), but always complete hair cell loss. Scale bars are 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384248&req=5

f2: ABR thresholds and histologic changes after unilateral neomycin deafening.(a) There was statistically significant elevation of thresholds overall (MANOVA, p < 0.01) and at each tested frequency (p < 0.05). Epi-fluorescence of auditory epithelium stained by phalloidin (actin, red) showed diverse tissue morphologies: scar formation (b), transition from scar formation to flat epithelium (c) or flat epithelium (d), but always complete hair cell loss. Scale bars are 25 μm.
Mentions: Clinically, candidate ears for stem cell therapy will have profound deafness due to a complete loss of hair cells. Therefore, we used deafened guinea pigs with widespread hair cell loss and profound deafness. We confirmed the outcome of the deafening insult using ABRs, measured one week after the unilateral deafening surgery. Statistically elevated ABR thresholds were obtained after deafening with neomycin (MANOVA; F = 174.79, df = 3, p < 0.001). Deafened animals had elevated thresholds at all frequencies (Fig. 2a). Residual hearing function was observed and probably is due to transmission of the sound stimulus to the undamaged (right) ear25. In the left ears, hair cells were invariably eliminated by the procedure. Supporting cells responded with differing degrees of pathological changes. In some areas, supporting cells remained in their normal height and exhibited the typical phalangeal scar formation (Fig. 2b). In other areas, we observed a transitional stage between scar formation and a flat epithelium (Fig. 2c), or flat epithelium (Fig. 2d).

View Article: PubMed Central - PubMed

ABSTRACT

Hair cells in the mature cochlea cannot spontaneously regenerate. One potential approach for restoring hair cells is stem cell therapy. However, when cells are transplanted into scala media (SM) of the cochlea, they promptly die due to the high potassium concentration. We previously described a method for conditioning the SM to make it more hospitable to implanted cells and showed that HeLa cells could survive for up to a week using this method. Here, we evaluated the survival of human embryonic stem cells (hESC) constitutively expressing GFP (H9 Cre-LoxP) in deaf guinea pig cochleae that were pre-conditioned to reduce potassium levels. GFP-positive cells could be detected in the cochlea for at least 7 days after the injection. The cells appeared spherical or irregularly shaped, and some were aggregated. Flushing SM with sodium caprate prior to transplantation resulted in a lower proportion of stem cells expressing the pluripotency marker Oct3/4 and increased cell survival. The data demonstrate that conditioning procedures aimed at transiently reducing the concentration of potassium in the SM facilitate survival of hESCs for at least one week. During this time window, additional procedures can be applied to initiate the differentiation of the implanted hESCs into new hair cells.

No MeSH data available.


Related in: MedlinePlus