Limits...
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.

Show MeSH

Related in: MedlinePlus

Differentiation of hAEC to progenitors of cortical neurons.(A) Representative micrographs at 20x from hAEC differentiated to Nestin-positive cells (green) after different treatments; the nuclei were stained with DAPI (blue). When the hAEC were treated with SB431542 + Noggin + EGF + bFGF the proportion of Nestin-positive cells increased. (B) Percentage of Nestin-positive cells obtained from hAEC during the proliferation stage with different treatments. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. (C) Representative micrographs of hAEC treated with SB431542 + Noggin + EGF + bFGF. On the 14th day, we found cells that were positive for OTX2 (red), TBR2 (green), NeuN (red), or β-III-tubulin (green); the nuclei were stained with DAPI (blue). Arrow indicate neurite prolongation. Scale bar 50 μm. * p < 0.05 as compared with basal medium (CM).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697857&req=5

pone.0146082.g006: Differentiation of hAEC to progenitors of cortical neurons.(A) Representative micrographs at 20x from hAEC differentiated to Nestin-positive cells (green) after different treatments; the nuclei were stained with DAPI (blue). When the hAEC were treated with SB431542 + Noggin + EGF + bFGF the proportion of Nestin-positive cells increased. (B) Percentage of Nestin-positive cells obtained from hAEC during the proliferation stage with different treatments. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. (C) Representative micrographs of hAEC treated with SB431542 + Noggin + EGF + bFGF. On the 14th day, we found cells that were positive for OTX2 (red), TBR2 (green), NeuN (red), or β-III-tubulin (green); the nuclei were stained with DAPI (blue). Arrow indicate neurite prolongation. Scale bar 50 μm. * p < 0.05 as compared with basal medium (CM).

Mentions: Finally, we differentiated hAEC to cortical progenitors. We tested 6 combinations of inhibitory molecules and growth factors in our cultures. At the first stage of the protocol, immunocytochemistry was performed to detect Nestin-positive cells after culturing in different conditions (Fig 6A). We found that the percentage of Nestin-positive cells increased significantly in medium containing bFGF+EGF+SB431542+Noggin versus control medium (Fig 6B). Cells from the bFGF+EGF+SB431542+Noggin condition were cultured for another 6 days without these molecules to induce cell differentiation. In these experiments we were able to identify cells expressing specific to progenitors of cortical neuron markers (OTX2, TBR2, β-III-tubulin, NeuN) by immunofluorescence (Fig 6C).


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)

Differentiation of hAEC to progenitors of cortical neurons.(A) Representative micrographs at 20x from hAEC differentiated to Nestin-positive cells (green) after different treatments; the nuclei were stained with DAPI (blue). When the hAEC were treated with SB431542 + Noggin + EGF + bFGF the proportion of Nestin-positive cells increased. (B) Percentage of Nestin-positive cells obtained from hAEC during the proliferation stage with different treatments. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. (C) Representative micrographs of hAEC treated with SB431542 + Noggin + EGF + bFGF. On the 14th day, we found cells that were positive for OTX2 (red), TBR2 (green), NeuN (red), or β-III-tubulin (green); the nuclei were stained with DAPI (blue). Arrow indicate neurite prolongation. Scale bar 50 μm. * p < 0.05 as compared with basal medium (CM).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146082.g006: Differentiation of hAEC to progenitors of cortical neurons.(A) Representative micrographs at 20x from hAEC differentiated to Nestin-positive cells (green) after different treatments; the nuclei were stained with DAPI (blue). When the hAEC were treated with SB431542 + Noggin + EGF + bFGF the proportion of Nestin-positive cells increased. (B) Percentage of Nestin-positive cells obtained from hAEC during the proliferation stage with different treatments. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. (C) Representative micrographs of hAEC treated with SB431542 + Noggin + EGF + bFGF. On the 14th day, we found cells that were positive for OTX2 (red), TBR2 (green), NeuN (red), or β-III-tubulin (green); the nuclei were stained with DAPI (blue). Arrow indicate neurite prolongation. Scale bar 50 μm. * p < 0.05 as compared with basal medium (CM).
Mentions: Finally, we differentiated hAEC to cortical progenitors. We tested 6 combinations of inhibitory molecules and growth factors in our cultures. At the first stage of the protocol, immunocytochemistry was performed to detect Nestin-positive cells after culturing in different conditions (Fig 6A). We found that the percentage of Nestin-positive cells increased significantly in medium containing bFGF+EGF+SB431542+Noggin versus control medium (Fig 6B). Cells from the bFGF+EGF+SB431542+Noggin condition were cultured for another 6 days without these molecules to induce cell differentiation. In these experiments we were able to identify cells expressing specific to progenitors of cortical neuron markers (OTX2, TBR2, β-III-tubulin, NeuN) by immunofluorescence (Fig 6C).

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