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

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.


The effect sodium caprate on density and stemness of transplanted hESCs.(a) Number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p < 0.05). (b) In the sodium caprate treated cochleae, the proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower. (c) A cochlea that was not flushed with sodium caprate, showing the high proportion of GFP-positive cells that were co-labelled with Oct3/4 in the nucleus. (d) A cochlea flushed with sodium caprate, showing a much smaller portion of GFP-positive cells co-labelled with Oct3/4. Scale bars are 25 μm.
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f6: The effect sodium caprate on density and stemness of transplanted hESCs.(a) Number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p < 0.05). (b) In the sodium caprate treated cochleae, the proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower. (c) A cochlea that was not flushed with sodium caprate, showing the high proportion of GFP-positive cells that were co-labelled with Oct3/4 in the nucleus. (d) A cochlea flushed with sodium caprate, showing a much smaller portion of GFP-positive cells co-labelled with Oct3/4. Scale bars are 25 μm.

Mentions: Sodium caprate has been shown to transiently disrupt the junctional complexes of epithelial cells1326. To enhance adhesion of the hESCs to the endogenous cells or facilitate insertion into the tissue, we added sodium caprate to the conditioning protocol. Transplantations were performed with or without sodium caprate, and animals were sacrificed at the 1-day time point. We then determined the density of the GFP-positive cells and assessed the proportion of Oct3/4-positive cells among GFP-positive cells for both treatment groups. The number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p = 0.020) (Fig. 6a). The proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower (p = 0.048) (Fig. 6b). In cochleae that were not flushed with sodium caprate, there were fewer GFP-positive cells and a higher proportion of them were co-labelled with Oct3/4 in the nucleus (Fig. 6c). This result indicates that including sodium caprate in the conditioning procedure increases survival of the GFP-positive cells at the 1-day time point. Some of the cells had very large nuclei (nearly 25 μm), which may indicate swelling as part of a degenerative process or another response to the high potassium concentration in the extracellular fluid of their new environment.


Survival of human embryonic stem cells implanted in the guinea pig auditory epithelium
The effect sodium caprate on density and stemness of transplanted hESCs.(a) Number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p < 0.05). (b) In the sodium caprate treated cochleae, the proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower. (c) A cochlea that was not flushed with sodium caprate, showing the high proportion of GFP-positive cells that were co-labelled with Oct3/4 in the nucleus. (d) A cochlea flushed with sodium caprate, showing a much smaller portion of GFP-positive cells co-labelled with Oct3/4. Scale bars are 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The effect sodium caprate on density and stemness of transplanted hESCs.(a) Number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p < 0.05). (b) In the sodium caprate treated cochleae, the proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower. (c) A cochlea that was not flushed with sodium caprate, showing the high proportion of GFP-positive cells that were co-labelled with Oct3/4 in the nucleus. (d) A cochlea flushed with sodium caprate, showing a much smaller portion of GFP-positive cells co-labelled with Oct3/4. Scale bars are 25 μm.
Mentions: Sodium caprate has been shown to transiently disrupt the junctional complexes of epithelial cells1326. To enhance adhesion of the hESCs to the endogenous cells or facilitate insertion into the tissue, we added sodium caprate to the conditioning protocol. Transplantations were performed with or without sodium caprate, and animals were sacrificed at the 1-day time point. We then determined the density of the GFP-positive cells and assessed the proportion of Oct3/4-positive cells among GFP-positive cells for both treatment groups. The number of GFP-positive cells was greater in cochleae flushed with sodium caprate (p = 0.020) (Fig. 6a). The proportion of GFP-positive cells expressing the stem cell marker Oct3/4 was significantly lower (p = 0.048) (Fig. 6b). In cochleae that were not flushed with sodium caprate, there were fewer GFP-positive cells and a higher proportion of them were co-labelled with Oct3/4 in the nucleus (Fig. 6c). This result indicates that including sodium caprate in the conditioning procedure increases survival of the GFP-positive cells at the 1-day time point. Some of the cells had very large nuclei (nearly 25 μm), which may indicate swelling as part of a degenerative process or another response to the high potassium concentration in the extracellular fluid of their new environment.

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.