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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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hAEC are positive for naïve pluripotent markers.(A) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) and TFE3 (red); the nuclei were stained with DAPI (blue). (B) Graph shows the percentage of hAEC that express naïve markers. (C) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) or TFE3 (red) and co-expressing TRA-1-60; the nuclei were stained with DAPI (blue). (D) Graph shows the percentage of cells immunostained for both TRA-1-60 and naïve markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. Scale bar 50 μm. * p < 0.05 as compared with P0.
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pone.0146082.g004: hAEC are positive for naïve pluripotent markers.(A) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) and TFE3 (red); the nuclei were stained with DAPI (blue). (B) Graph shows the percentage of hAEC that express naïve markers. (C) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) or TFE3 (red) and co-expressing TRA-1-60; the nuclei were stained with DAPI (blue). (D) Graph shows the percentage of cells immunostained for both TRA-1-60 and naïve markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. Scale bar 50 μm. * p < 0.05 as compared with P0.

Mentions: Once we determined the expression of pluripotent markers in our conditions, we tested for the presence of the naïve pluripotent markers, KLF4 and TFE3. Interestingly, these transcription factors were present in the hAEC from P0-P2, as shown by immunofluorescence (Fig 4A). Our results indicated a significant increase in the percentage of cells that express KLF4 and TFE3 in P2 in comparison with P0 (Fig 4B). We evaluated if KLF4- and TFE3-positive hAEC co-expressed TRA-1-60 (Fig 4C). The number of double-labeled cells showed that only ~ 2% of the cells were positive for both KLF4-TRA-1-60 and TFE3-TRA-1-60 at the end of P2 (Fig 4D).


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 are positive for naïve pluripotent markers.(A) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) and TFE3 (red); the nuclei were stained with DAPI (blue). (B) Graph shows the percentage of hAEC that express naïve markers. (C) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) or TFE3 (red) and co-expressing TRA-1-60; the nuclei were stained with DAPI (blue). (D) Graph shows the percentage of cells immunostained for both TRA-1-60 and naïve markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. Scale bar 50 μm. * p < 0.05 as compared with P0.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146082.g004: hAEC are positive for naïve pluripotent markers.(A) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) and TFE3 (red); the nuclei were stained with DAPI (blue). (B) Graph shows the percentage of hAEC that express naïve markers. (C) Representative micrographs at 20x from hAEC at P0-P2 immunostained for KLF4 (red) or TFE3 (red) and co-expressing TRA-1-60; the nuclei were stained with DAPI (blue). (D) Graph shows the percentage of cells immunostained for both TRA-1-60 and naïve markers. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. Scale bar 50 μm. * p < 0.05 as compared with P0.
Mentions: Once we determined the expression of pluripotent markers in our conditions, we tested for the presence of the naïve pluripotent markers, KLF4 and TFE3. Interestingly, these transcription factors were present in the hAEC from P0-P2, as shown by immunofluorescence (Fig 4A). Our results indicated a significant increase in the percentage of cells that express KLF4 and TFE3 in P2 in comparison with P0 (Fig 4B). We evaluated if KLF4- and TFE3-positive hAEC co-expressed TRA-1-60 (Fig 4C). The number of double-labeled cells showed that only ~ 2% of the cells were positive for both KLF4-TRA-1-60 and TFE3-TRA-1-60 at the end of P2 (Fig 4D).

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