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

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.

Fig 1. 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).

Fig 2. 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.

Fig 3. Proliferation of hAEC that display pluripotent markers in vitro.

(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) and also for Ki67 (green or red); the nuclei were stained with DAPI (blue). (B) Graph shows the percentage of hAEC positive for one of the pluripotency factors and Ki67. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from five independent experiments. Scale bar 50 μm.

Fig 4. 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.

Fig 5. Epigenetic state of hAEC.

(A) Representative micrographs at 20x from hAEC at P0-P2 immunostained for H3K27me3 (red); the nuclei were stained with DAPI (blue). (B) Graph showing the percentage of hAEC that present the H3K27me3 mark. (C) Representative micrographs at 20x from hAEC at P0-P2 immunostained for H3K24me3 (green); the nuclei were stained with DAPI (blue). (D) Graph showing the percentage of hAEC that present the H3K4me3 mark. (E) Representative micrographs at 20x from hAEC at P0-P2 immunostained for H3K27me3 (red) and Ki67 (green); the nuclei were stained with DAPI (blue). (F) Percentage of hAEC positive for both H3K27me3 and Ki67. Results are expressed as percentages of means ± S.E.M. from 9 fields counted in duplicate from three independent experiments. Scale bars, 50 μm.

Fig 6. 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 14^th 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).