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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 cultured in vitro express genes associated with pluripotency.(A) Representative images of the electrophoresis of RT-PCR products of mRNAs for transcription factors OCT4 (151 bp), SOX2 (264 bp), NANOG (286 bp), REX1 (306 bp), KLF4 (134 bp) of hAEC cultured in vitro. L = ladder, H9 = hESC line H9 (positive control), P = passage. As negative control (-RT), the reverse transcriptase enzyme was not added. (B) Relative expression levels obtained from RT-qPCR of the pluripotency genes (mean from 5 independent experiments).
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pone.0146082.g001: hAEC cultured in vitro express genes associated with pluripotency.(A) Representative images of the electrophoresis of RT-PCR products of mRNAs for transcription factors OCT4 (151 bp), SOX2 (264 bp), NANOG (286 bp), REX1 (306 bp), KLF4 (134 bp) of hAEC cultured in vitro. L = ladder, H9 = hESC line H9 (positive control), P = passage. As negative control (-RT), the reverse transcriptase enzyme was not added. (B) Relative expression levels obtained from RT-qPCR of the pluripotency genes (mean from 5 independent experiments).

Mentions: Expression of OCT4, SOX2, NANOG, REX1 and KLF4 in H9 and hAEC from P0-P3 was analyzed qualitatively by RT-PCR. Our results showed that hAEC express all of these transcription factors in all passages, and the amplified products were of the predicted size (Fig 1A and S2 Fig). As expected, the hESC positive control also expressed these markers.


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 cultured in vitro express genes associated with pluripotency.(A) Representative images of the electrophoresis of RT-PCR products of mRNAs for transcription factors OCT4 (151 bp), SOX2 (264 bp), NANOG (286 bp), REX1 (306 bp), KLF4 (134 bp) of hAEC cultured in vitro. L = ladder, H9 = hESC line H9 (positive control), P = passage. As negative control (-RT), the reverse transcriptase enzyme was not added. (B) Relative expression levels obtained from RT-qPCR of the pluripotency genes (mean from 5 independent experiments).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146082.g001: hAEC cultured in vitro express genes associated with pluripotency.(A) Representative images of the electrophoresis of RT-PCR products of mRNAs for transcription factors OCT4 (151 bp), SOX2 (264 bp), NANOG (286 bp), REX1 (306 bp), KLF4 (134 bp) of hAEC cultured in vitro. L = ladder, H9 = hESC line H9 (positive control), P = passage. As negative control (-RT), the reverse transcriptase enzyme was not added. (B) Relative expression levels obtained from RT-qPCR of the pluripotency genes (mean from 5 independent experiments).
Mentions: Expression of OCT4, SOX2, NANOG, REX1 and KLF4 in H9 and hAEC from P0-P3 was analyzed qualitatively by RT-PCR. Our results showed that hAEC express all of these transcription factors in all passages, and the amplified products were of the predicted size (Fig 1A and S2 Fig). As expected, the hESC positive control also expressed these markers.

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