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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

Schematic representation of the HC programming strategy used to generate and purify iHCs in vitro at an early (day 8) and later stage (day 12) of their development. FACS, fluorescence activated cell sorting.
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f0005: Schematic representation of the HC programming strategy used to generate and purify iHCs in vitro at an early (day 8) and later stage (day 12) of their development. FACS, fluorescence activated cell sorting.

Mentions: The embryonic stem cell (ESC) line iGPA-Myo7a:mVenus used in this study was generated from the Ainv15 ESC line [1] by two genetic engineering steps: I) a DNA fragment containing the three genes Gfi1-Pou4f3-Atoh1 (GPA) was inserted into the doxycycline (dox) inducible locus of the Ainv15 cells as previously described in [2] to produce the iGPA line. II) The mVenus fluorescent reporter under control of the promoter and regulatory regions of the incipient HC marker, Myo7a [3] was randomly inserted into the iGPA genome as described in [4], to generate the iGPA-Myo7a:mVenus line. These ESCs were routinely grown on gelatin-coated (0.1%) Nunc dishes at 37 °C in a 5% CO2 incubator with Dulbecco's Modified Eagles Medium 1 × (DMEM, GIBCO) supplemented with 10% of heat-inactivated fetal bovine serum (FBS) (GIBCO, ES-qualified), 2 mM glutamine (GIBCO), 1% penicillin/streptomycin (GIBCO), 1 mM sodium pyruvate (GIBCO), 1% MEM non-essential amino acids (GIBCO), 2 ng/ml Leukemia inhibitory factor (LIF) and 7 μm 2-mercaptoethanol (Sigma). All solutions were mixed and filtered through a 0.2 μm filter unit into a new sterile flask. Cells were passaged every other day, at constant plating density of 3 × 104 cells/cm2. To generate embryoid bodies (EBs), ESCs were dissociated into single cells (using 0025% trypsin at 37 °C for 3 min) and plated at low density (2 × 104 cells/cm2) with the same supplemented DMEM medium but without LIF, in 10 cm bacterial grade petri dishes to prevent attachment. EB formation was checked on day 1 and medium was replaced every two days (days 2, 4, 6, 8 and 10). Supplementation with 2 μg/ml doxycycline (Sigma, diluted in sterile PBS and filtered through a 0.2 μm filter unit), 1 μm retinoic acid (RA) (diluted in 0.01% DMSO, Sigma) was initiated at day 4 and maintained until the required time point for analysis (day 8 or day 12) (Fig. 1).


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)

Schematic representation of the HC programming strategy used to generate and purify iHCs in vitro at an early (day 8) and later stage (day 12) of their development. FACS, fluorescence activated cell sorting.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: Schematic representation of the HC programming strategy used to generate and purify iHCs in vitro at an early (day 8) and later stage (day 12) of their development. FACS, fluorescence activated cell sorting.
Mentions: The embryonic stem cell (ESC) line iGPA-Myo7a:mVenus used in this study was generated from the Ainv15 ESC line [1] by two genetic engineering steps: I) a DNA fragment containing the three genes Gfi1-Pou4f3-Atoh1 (GPA) was inserted into the doxycycline (dox) inducible locus of the Ainv15 cells as previously described in [2] to produce the iGPA line. II) The mVenus fluorescent reporter under control of the promoter and regulatory regions of the incipient HC marker, Myo7a [3] was randomly inserted into the iGPA genome as described in [4], to generate the iGPA-Myo7a:mVenus line. These ESCs were routinely grown on gelatin-coated (0.1%) Nunc dishes at 37 °C in a 5% CO2 incubator with Dulbecco's Modified Eagles Medium 1 × (DMEM, GIBCO) supplemented with 10% of heat-inactivated fetal bovine serum (FBS) (GIBCO, ES-qualified), 2 mM glutamine (GIBCO), 1% penicillin/streptomycin (GIBCO), 1 mM sodium pyruvate (GIBCO), 1% MEM non-essential amino acids (GIBCO), 2 ng/ml Leukemia inhibitory factor (LIF) and 7 μm 2-mercaptoethanol (Sigma). All solutions were mixed and filtered through a 0.2 μm filter unit into a new sterile flask. Cells were passaged every other day, at constant plating density of 3 × 104 cells/cm2. To generate embryoid bodies (EBs), ESCs were dissociated into single cells (using 0025% trypsin at 37 °C for 3 min) and plated at low density (2 × 104 cells/cm2) with the same supplemented DMEM medium but without LIF, in 10 cm bacterial grade petri dishes to prevent attachment. EB formation was checked on day 1 and medium was replaced every two days (days 2, 4, 6, 8 and 10). Supplementation with 2 μg/ml doxycycline (Sigma, diluted in sterile PBS and filtered through a 0.2 μm filter unit), 1 μm retinoic acid (RA) (diluted in 0.01% DMSO, Sigma) was initiated at day 4 and maintained until the required time point for analysis (day 8 or day 12) (Fig. 1).

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