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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 PAX2 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) Pax2 is expressed in otic placodal cells during the early stages of AN development. (B) Using immunostaining, an increase in the expression of PAX2 was observed at 35 DIV. Scale bar=100 μm (relative to all images). (C-I) There was a significant increase in the percentages of hiPSC and hESC neurospheres that had PAX2 immunopositive cells. Data obtained from at least three independent experiments and expressed as means±SEM of triplicates of each sample. (C-II) The qRT-PCR data show the fold changes of PAX2 in the cell lines at 19 and 35 DIV. The results were presented as fold changes relative to the endogenous control, ß-ACTIN, and the undifferentiated stem cells. Each point on the graph represents mean±SD (n=3). *p<0.05; **p<0.01; ***p<0.001. DIV, days in vitro; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; SEM, standard error of the mean.
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f2: Expression of PAX2 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) Pax2 is expressed in otic placodal cells during the early stages of AN development. (B) Using immunostaining, an increase in the expression of PAX2 was observed at 35 DIV. Scale bar=100 μm (relative to all images). (C-I) There was a significant increase in the percentages of hiPSC and hESC neurospheres that had PAX2 immunopositive cells. Data obtained from at least three independent experiments and expressed as means±SEM of triplicates of each sample. (C-II) The qRT-PCR data show the fold changes of PAX2 in the cell lines at 19 and 35 DIV. The results were presented as fold changes relative to the endogenous control, ß-ACTIN, and the undifferentiated stem cells. Each point on the graph represents mean±SD (n=3). *p<0.05; **p<0.01; ***p<0.001. DIV, days in vitro; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; SEM, standard error of the mean.

Mentions: To investigate whether the hiPSC and hESC neural progenitors derived from this assay were differentiating toward an otic placodal lineage, the expression of PAX2 was also examined (Fig. 2A). Immunocytochemical results indicated that there was a significant increase in the percentages of neurospheres that had PAX2 positive cells at 35 DIV [compared with 19 DIV; iPS1(A) and H9: p<0.001, iPS1(B): p<0.01, and iPS2: p<0.05; Fig. 2B, C-I]. Notably, at 35 DIV, all neurospheres examined contained PAX2-positive cells. However, variability in the levels of PAX2 expression was noticed between the hiPSC lines (iPS1 and iPS2), between samples of the same hiPSC line [iPS1(A) and iPS1(B)], and between the hiPSCs and hESCs. To quantify the expression levels of PAX2, qRT-PCR was employed. Consistent with the immunocytochemical data, the qRT-PCR results revealed an upregulation of PAX2 in the hiPSC and hESC-derived neurosensory progenitors at 19 DIV (iPS2: p<0.05; H9: p<0.001) and 35 DIV [when compared with the undifferentiated controls; iPS1(B) and H9: p<0.05; Fig. 2C-II]. Although variabilities in the mean fold changes of PAX2 were also observed between the hiPSC lines, samples of the same hiPSC line, and between the hiPSC and hESC lines, collectively, these findings indicated that PAX2 mRNA and protein could be detected in both the hiPSC- and hESC-derived neural progenitors across different time points during 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 PAX2 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) Pax2 is expressed in otic placodal cells during the early stages of AN development. (B) Using immunostaining, an increase in the expression of PAX2 was observed at 35 DIV. Scale bar=100 μm (relative to all images). (C-I) There was a significant increase in the percentages of hiPSC and hESC neurospheres that had PAX2 immunopositive cells. Data obtained from at least three independent experiments and expressed as means±SEM of triplicates of each sample. (C-II) The qRT-PCR data show the fold changes of PAX2 in the cell lines at 19 and 35 DIV. The results were presented as fold changes relative to the endogenous control, ß-ACTIN, and the undifferentiated stem cells. Each point on the graph represents mean±SD (n=3). *p<0.05; **p<0.01; ***p<0.001. DIV, days in vitro; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; SEM, standard error of the mean.
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Related In: Results  -  Collection

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f2: Expression of PAX2 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) Pax2 is expressed in otic placodal cells during the early stages of AN development. (B) Using immunostaining, an increase in the expression of PAX2 was observed at 35 DIV. Scale bar=100 μm (relative to all images). (C-I) There was a significant increase in the percentages of hiPSC and hESC neurospheres that had PAX2 immunopositive cells. Data obtained from at least three independent experiments and expressed as means±SEM of triplicates of each sample. (C-II) The qRT-PCR data show the fold changes of PAX2 in the cell lines at 19 and 35 DIV. The results were presented as fold changes relative to the endogenous control, ß-ACTIN, and the undifferentiated stem cells. Each point on the graph represents mean±SD (n=3). *p<0.05; **p<0.01; ***p<0.001. DIV, days in vitro; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; SEM, standard error of the mean.
Mentions: To investigate whether the hiPSC and hESC neural progenitors derived from this assay were differentiating toward an otic placodal lineage, the expression of PAX2 was also examined (Fig. 2A). Immunocytochemical results indicated that there was a significant increase in the percentages of neurospheres that had PAX2 positive cells at 35 DIV [compared with 19 DIV; iPS1(A) and H9: p<0.001, iPS1(B): p<0.01, and iPS2: p<0.05; Fig. 2B, C-I]. Notably, at 35 DIV, all neurospheres examined contained PAX2-positive cells. However, variability in the levels of PAX2 expression was noticed between the hiPSC lines (iPS1 and iPS2), between samples of the same hiPSC line [iPS1(A) and iPS1(B)], and between the hiPSCs and hESCs. To quantify the expression levels of PAX2, qRT-PCR was employed. Consistent with the immunocytochemical data, the qRT-PCR results revealed an upregulation of PAX2 in the hiPSC and hESC-derived neurosensory progenitors at 19 DIV (iPS2: p<0.05; H9: p<0.001) and 35 DIV [when compared with the undifferentiated controls; iPS1(B) and H9: p<0.05; Fig. 2C-II]. Although variabilities in the mean fold changes of PAX2 were also observed between the hiPSC lines, samples of the same hiPSC line, and between the hiPSC and hESC lines, collectively, these findings indicated that PAX2 mRNA and protein could be detected in both the hiPSC- and hESC-derived neural progenitors across different time points during 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