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Directing human induced pluripotent stem cells into a neurosensory lineage for auditory neuron replacement.

Gunewardene N, Bergen NV, Crombie D, Needham K, Dottori M, Nayagam BA - Biores Open Access (2014)

Bottom Line: Furthermore, the neurons generated from this assay were found to be electrically active.While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls.Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology, University of Melbourne , East Melbourne, Victoria, Australia .

ABSTRACT
Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

No MeSH data available.


Related in: MedlinePlus

Expression of SOX2 and BRN3A in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) SOX2 is expressed during the proneurosensory stage of AN development. (B) SOX2 immunopositive cells were observed in the hiPSC and hESC neurospheres at all the time points analyzed. (C) The box inserts of the merged images of (B) show that at 19 DIV and 35 DIV, there were some cells that were BRN3A+/SOX2+ (arrows); however, the majority were either BRN3A+/SOX2− and/or BRN3A−/SOX2+. Scale bar=100μm (all images).
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f3: Expression of SOX2 and BRN3A in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) SOX2 is expressed during the proneurosensory stage of AN development. (B) SOX2 immunopositive cells were observed in the hiPSC and hESC neurospheres at all the time points analyzed. (C) The box inserts of the merged images of (B) show that at 19 DIV and 35 DIV, there were some cells that were BRN3A+/SOX2+ (arrows); however, the majority were either BRN3A+/SOX2− and/or BRN3A−/SOX2+. Scale bar=100μm (all images).

Mentions: In addition to Pax2, the transcription factor Sox2 has been found to play a critical role during early AN development in promoting otic epithelial cells into a proneurosensory lineage (Fig. 3A).21,22 The coexpression of SOX2 with the sensory neural marker BRN3A was analyzed in all neurospheres at 19 and 35 DIV (Fig. 3B). We found that SOX2 was expressed in all the hiPSC and hESC neurospheres analyzed at 19 and 35 DIV. Coimmunostaining with both SOX2 and BRN3A showed subpopulations of cells that were BRN3A+/SOX2+, BRN3A−/SOX+, or BRN3A+/SOX2− (Fig. 3C). This may represent different progenitor subpopulations and/or progenitors that are at different temporal stages of differentiation.


Directing human induced pluripotent stem cells into a neurosensory lineage for auditory neuron replacement.

Gunewardene N, Bergen NV, Crombie D, Needham K, Dottori M, Nayagam BA - Biores Open Access (2014)

Expression of SOX2 and BRN3A in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) SOX2 is expressed during the proneurosensory stage of AN development. (B) SOX2 immunopositive cells were observed in the hiPSC and hESC neurospheres at all the time points analyzed. (C) The box inserts of the merged images of (B) show that at 19 DIV and 35 DIV, there were some cells that were BRN3A+/SOX2+ (arrows); however, the majority were either BRN3A+/SOX2− and/or BRN3A−/SOX2+. Scale bar=100μm (all images).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4120935&req=5

f3: Expression of SOX2 and BRN3A in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) SOX2 is expressed during the proneurosensory stage of AN development. (B) SOX2 immunopositive cells were observed in the hiPSC and hESC neurospheres at all the time points analyzed. (C) The box inserts of the merged images of (B) show that at 19 DIV and 35 DIV, there were some cells that were BRN3A+/SOX2+ (arrows); however, the majority were either BRN3A+/SOX2− and/or BRN3A−/SOX2+. Scale bar=100μm (all images).
Mentions: In addition to Pax2, the transcription factor Sox2 has been found to play a critical role during early AN development in promoting otic epithelial cells into a proneurosensory lineage (Fig. 3A).21,22 The coexpression of SOX2 with the sensory neural marker BRN3A was analyzed in all neurospheres at 19 and 35 DIV (Fig. 3B). We found that SOX2 was expressed in all the hiPSC and hESC neurospheres analyzed at 19 and 35 DIV. Coimmunostaining with both SOX2 and BRN3A showed subpopulations of cells that were BRN3A+/SOX2+, BRN3A−/SOX+, or BRN3A+/SOX2− (Fig. 3C). This may represent different progenitor subpopulations and/or progenitors that are at different temporal stages of differentiation.

Bottom Line: Furthermore, the neurons generated from this assay were found to be electrically active.While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls.Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology, University of Melbourne , East Melbourne, Victoria, Australia .

ABSTRACT
Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

No MeSH data available.


Related in: MedlinePlus