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Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.

García-Castro IL, García-López G, Ávila-González D, Flores-Herrera H, Molina-Hernández A, Portillo W, Ramón-Gallegos E, Díaz NF - PLoS ONE (2015)

Bottom Line: Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin).Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation.This highlights the need for further investigation of hAEC as a possible source of hPSC.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Citopatología Ambiental, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Campus Zacatenco, Unidad Profesional "Adolfo López Mateos", México D.F., México.

ABSTRACT
Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

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Related in: MedlinePlus

hAEC display the pluripotent stem cell markers.(A) Representative micrographs at 20X from hAEC at different passages (P0-P3) immunostained for OCT4 (red), SOX2 (red), NANOG (red), SSEA3 (green), SSEA4 (green), TRA-1-60 (green) and E-cadherin (green); the nuclei were stained with DAPI (blue). Arrow indicate that the marker was found in the cytoplasm. (B) Graph shows the percentage of hAEC positive for the different pluripotent markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from five independent experiments. Scale bar 50 μm.
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pone.0146082.g002: hAEC display the pluripotent stem cell markers.(A) Representative micrographs at 20X from hAEC at different passages (P0-P3) immunostained for OCT4 (red), SOX2 (red), NANOG (red), SSEA3 (green), SSEA4 (green), TRA-1-60 (green) and E-cadherin (green); the nuclei were stained with DAPI (blue). Arrow indicate that the marker was found in the cytoplasm. (B) Graph shows the percentage of hAEC positive for the different pluripotent markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from five independent experiments. Scale bar 50 μm.

Mentions: The pluripotent markers OCT4, SOX2 and NANOG were detected in hAEC through passages P0-P3. Interestingly, in early passages, the markers were present in the cells' cytoplasmatic membrane and nucleus. However, in P3 the immunostaining was localized exclusively in the nucleus (Fig 2A). The percentage of hAEC that expressed OCT4, SOX2 and NANOG during the various passages did not change significantly (Fig 2B).


Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.

García-Castro IL, García-López G, Ávila-González D, Flores-Herrera H, Molina-Hernández A, Portillo W, Ramón-Gallegos E, Díaz NF - PLoS ONE (2015)

hAEC display the pluripotent stem cell markers.(A) Representative micrographs at 20X from hAEC at different passages (P0-P3) immunostained for OCT4 (red), SOX2 (red), NANOG (red), SSEA3 (green), SSEA4 (green), TRA-1-60 (green) and E-cadherin (green); the nuclei were stained with DAPI (blue). Arrow indicate that the marker was found in the cytoplasm. (B) Graph shows the percentage of hAEC positive for the different pluripotent markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from five independent experiments. Scale bar 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697857&req=5

pone.0146082.g002: hAEC display the pluripotent stem cell markers.(A) Representative micrographs at 20X from hAEC at different passages (P0-P3) immunostained for OCT4 (red), SOX2 (red), NANOG (red), SSEA3 (green), SSEA4 (green), TRA-1-60 (green) and E-cadherin (green); the nuclei were stained with DAPI (blue). Arrow indicate that the marker was found in the cytoplasm. (B) Graph shows the percentage of hAEC positive for the different pluripotent markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from five independent experiments. Scale bar 50 μm.
Mentions: The pluripotent markers OCT4, SOX2 and NANOG were detected in hAEC through passages P0-P3. Interestingly, in early passages, the markers were present in the cells' cytoplasmatic membrane and nucleus. However, in P3 the immunostaining was localized exclusively in the nucleus (Fig 2A). The percentage of hAEC that expressed OCT4, SOX2 and NANOG during the various passages did not change significantly (Fig 2B).

Bottom Line: Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin).Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation.This highlights the need for further investigation of hAEC as a possible source of hPSC.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Citopatología Ambiental, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Campus Zacatenco, Unidad Profesional "Adolfo López Mateos", México D.F., México.

ABSTRACT
Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

Show MeSH
Related in: MedlinePlus