Limits...
Transcriptome profiling of induced hair cells (iHCs) generated by combined expression of Gfi1, Pou4f3 and Atoh1 during embryonic stem cell differentiation.

Costa A, Henrique D - Genom Data (2015)

Bottom Line: We found that combined activity of three transcription factors, Gfi1, Pou4f3, and Atoh1, can program ESC-derived progenitors towards HC fate with efficiencies of 55%-80%.This approach based on FACS sorting and microarray analysis revealed a highly similar iHC transcriptome to that of endogenous HCs in vivo.The data obtained in this study is available in the Gene Expression Omnibus (GEO) database (accession number GSE60352).

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa 1649-028, Portugal.

ABSTRACT
To gain new insights about the genetic networks controlling hair cell (HC) development, we previously developed a direct genetic programming strategy to generate an inexhaustible supply of HC-like cells (induced HCs, iHCs) in vitro, starting from mouse embryonic stem cells (ESC). We found that combined activity of three transcription factors, Gfi1, Pou4f3, and Atoh1, can program ESC-derived progenitors towards HC fate with efficiencies of 55%-80%. These iHCs express several HC markers and exhibit polarized structures that are highly reminiscent of the mechanosensitive hair bundles, with many microvilli-like stereocilia. Here, we describe the experimental design, methodology, and data validation for the microarray analysis used to characterize the transcriptome profile of iHCs at different stages of their differentiation. This approach based on FACS sorting and microarray analysis revealed a highly similar iHC transcriptome to that of endogenous HCs in vivo. The data obtained in this study is available in the Gene Expression Omnibus (GEO) database (accession number GSE60352).

No MeSH data available.


Related in: MedlinePlus

Relative mRNA levels measured by qPCR for Venus and HC markers (Myo7a, Myo6, Espin and Cdh23) in the mVenus fluorescent reporter venus-positive (green) and venus-negative (black) sorted populations obtained from EBs treated with Dox or Dox + RA at day 8 and at day 12. Relative expression of each transcript is presented as fold change normalized to the mean of untreated EBs (arbitrary set to 1). Unpaired t-test was used for statistical analysis. *P < 0.05 **P < 0.01 ***P < 0.001 (n = 3).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664721&req=5

f0010: Relative mRNA levels measured by qPCR for Venus and HC markers (Myo7a, Myo6, Espin and Cdh23) in the mVenus fluorescent reporter venus-positive (green) and venus-negative (black) sorted populations obtained from EBs treated with Dox or Dox + RA at day 8 and at day 12. Relative expression of each transcript is presented as fold change normalized to the mean of untreated EBs (arbitrary set to 1). Unpaired t-test was used for statistical analysis. *P < 0.05 **P < 0.01 ***P < 0.001 (n = 3).

Mentions: Sorted Venus-positive cell populations from EBs were collected with high purity (~ 98%) and the Venus-negative cell fraction was discarded for the microarray analysis. However, these nonfluorescent cells were used for quantitative-real time PCR (qPCR) analysis, to validate samples and to determine the enrichment of the iHC population in the Venus-positive fraction. The qPCR reaction was performed on 96-well plates (MicroAmp; Applied Biosystems) or 384-well plates (MicroAmp; Applied Biosystems) covered with optical adhesive covers (Applied Biosystems). The instruments used were Applied Biosystems 7500 Real-Time PCR or Applied Biosystems ViiA 7 Real-Time PCR. The Real-Time PCR was carried out using iTaq Universal Sybr Green Supermix (Bio-Rad), 2 μl of the retrotranscription cDNA template diluted 1:100 and 12,5 pmol of each primer. Reaction conditions were as follows: one step of 50 °C for 2 min, one 95 °C for 10 min, and 40 cycles of 95 °C for 15 s denaturation and 60 °C for 1 min annealing and extension. The cDNA was used as template for each pair of primers in a duplicate PCR reaction. GAPDH was used was a calibrator. Relative expression levels in the various Dox-treated samples were referred to the levels of expression in untreated control (without Dox), which were arbitrarily set to 1. Results are shown as averages ± standard error of mean (SEM) of three independent experiments. As expected, strong Venus expression was only observed among the sorted Venus-positive cells (Fig. 2). Myo7a expression was significantly higher in the Venus-positive sorted cells and HC markers such as Myo6[5], Espin[6], and Cdh23[7] were also enriched in this fraction (Fig. 2). Overall, the results confirmed the high degree of purity of the iHC-sorted cells.


Transcriptome profiling of induced hair cells (iHCs) generated by combined expression of Gfi1, Pou4f3 and Atoh1 during embryonic stem cell differentiation.

Costa A, Henrique D - Genom Data (2015)

Relative mRNA levels measured by qPCR for Venus and HC markers (Myo7a, Myo6, Espin and Cdh23) in the mVenus fluorescent reporter venus-positive (green) and venus-negative (black) sorted populations obtained from EBs treated with Dox or Dox + RA at day 8 and at day 12. Relative expression of each transcript is presented as fold change normalized to the mean of untreated EBs (arbitrary set to 1). Unpaired t-test was used for statistical analysis. *P < 0.05 **P < 0.01 ***P < 0.001 (n = 3).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664721&req=5

f0010: Relative mRNA levels measured by qPCR for Venus and HC markers (Myo7a, Myo6, Espin and Cdh23) in the mVenus fluorescent reporter venus-positive (green) and venus-negative (black) sorted populations obtained from EBs treated with Dox or Dox + RA at day 8 and at day 12. Relative expression of each transcript is presented as fold change normalized to the mean of untreated EBs (arbitrary set to 1). Unpaired t-test was used for statistical analysis. *P < 0.05 **P < 0.01 ***P < 0.001 (n = 3).
Mentions: Sorted Venus-positive cell populations from EBs were collected with high purity (~ 98%) and the Venus-negative cell fraction was discarded for the microarray analysis. However, these nonfluorescent cells were used for quantitative-real time PCR (qPCR) analysis, to validate samples and to determine the enrichment of the iHC population in the Venus-positive fraction. The qPCR reaction was performed on 96-well plates (MicroAmp; Applied Biosystems) or 384-well plates (MicroAmp; Applied Biosystems) covered with optical adhesive covers (Applied Biosystems). The instruments used were Applied Biosystems 7500 Real-Time PCR or Applied Biosystems ViiA 7 Real-Time PCR. The Real-Time PCR was carried out using iTaq Universal Sybr Green Supermix (Bio-Rad), 2 μl of the retrotranscription cDNA template diluted 1:100 and 12,5 pmol of each primer. Reaction conditions were as follows: one step of 50 °C for 2 min, one 95 °C for 10 min, and 40 cycles of 95 °C for 15 s denaturation and 60 °C for 1 min annealing and extension. The cDNA was used as template for each pair of primers in a duplicate PCR reaction. GAPDH was used was a calibrator. Relative expression levels in the various Dox-treated samples were referred to the levels of expression in untreated control (without Dox), which were arbitrarily set to 1. Results are shown as averages ± standard error of mean (SEM) of three independent experiments. As expected, strong Venus expression was only observed among the sorted Venus-positive cells (Fig. 2). Myo7a expression was significantly higher in the Venus-positive sorted cells and HC markers such as Myo6[5], Espin[6], and Cdh23[7] were also enriched in this fraction (Fig. 2). Overall, the results confirmed the high degree of purity of the iHC-sorted cells.

Bottom Line: We found that combined activity of three transcription factors, Gfi1, Pou4f3, and Atoh1, can program ESC-derived progenitors towards HC fate with efficiencies of 55%-80%.This approach based on FACS sorting and microarray analysis revealed a highly similar iHC transcriptome to that of endogenous HCs in vivo.The data obtained in this study is available in the Gene Expression Omnibus (GEO) database (accession number GSE60352).

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa 1649-028, Portugal.

ABSTRACT
To gain new insights about the genetic networks controlling hair cell (HC) development, we previously developed a direct genetic programming strategy to generate an inexhaustible supply of HC-like cells (induced HCs, iHCs) in vitro, starting from mouse embryonic stem cells (ESC). We found that combined activity of three transcription factors, Gfi1, Pou4f3, and Atoh1, can program ESC-derived progenitors towards HC fate with efficiencies of 55%-80%. These iHCs express several HC markers and exhibit polarized structures that are highly reminiscent of the mechanosensitive hair bundles, with many microvilli-like stereocilia. Here, we describe the experimental design, methodology, and data validation for the microarray analysis used to characterize the transcriptome profile of iHCs at different stages of their differentiation. This approach based on FACS sorting and microarray analysis revealed a highly similar iHC transcriptome to that of endogenous HCs in vivo. The data obtained in this study is available in the Gene Expression Omnibus (GEO) database (accession number GSE60352).

No MeSH data available.


Related in: MedlinePlus