Progesterone receptor is constitutively expressed in induced Pluripotent Stem Cells (iPSCs)

Abstract

Induced Pluripotent Stem Cells (iPSCs) are nowadays a common starting point for wide-ranging applications including 3D disease modeling (i.e. organoids) and in future regenerative medicine. Physiological processes like homeostasis, cell differentiation, development and reproduction are tightly regulated by hormones through binding to their transmembrane or nuclear receptors of target cells. Considering their pleiotropic effect, take into account also their expression in an iPSCs-based disease modeling would better recapitulate the molecular events leading to 3D organoid development and disease study. Here we reported the expression pattern of estrogen receptor (ERα) and progesterone receptor (PR) in four different iPSCs, obtained from CD34 + progenitor cells and skin fibroblasts with four different methods. Expression of ERα and PR mRNA were significantly downregulated in iPSCs as well as fibroblasts compared to MCF7 positive control. Immunofluorescence (IF) staining detected only the expression of PR protein in all the different iPSCs cell lines, while ERα was not detectable. By flow cytometry analysis we observed that the ~ 65% of the total population of iPSCs cells expressed only PR, with 100% fold increase compared to HSPCs and fibroblasts, while ERα was not expressed. Our results collectively demonstrated for the first time that the reprogramming of somatic cells into iPSCs leads to the expression of PR receptor.

Keywords: CD34; Differentiation; Estrogen receptor; Fibroblasts; Induced pluripotent stem cells (iPSCs); Progesterone receptor.

Fig. 1

Expression profile of ERα/β and PR receptors in iPSCs compared to MCF7 and MDA-MB-231. A ERα mRNA in iPSCs cell lines compared to MCF7 positive and MDA-MB-231 negative control. B ERβ mRNA in iPSCs cell lines compared to MCF7 negative and MDA-MB-231 positive control. C PR mRNA in iPSCs cell lines compared to MCF7 positive and MDA-MB-231 negative control. Histograms represent fold-change in the gene expression of three independent experiments, while error bars represent ± SEM. One-way ANOVA followed by Tukey’s post-hoc test. **** p < 0.0001

Fig. 2

Detection of ERα in iPSCs cell lines. Immunofluorescent (IF) staining for the detection of ERα among the different iPSCs cell lines. Nuclei were counterstained in blue (DAPI), while cytoskeleton in green (phalloidin-488) and ERα in red (Alexa-568). Magnification 10X

Fig. 3

Detection of PR in iPSCs cell lines. Immunofluorescent (IF) staining for the detection of PR among the different iPSCs cell lines. Nuclei were counterstained in blue (DAPI), while cytoskeleton in green (phalloidin-488) and PR in red (Alexa-568). Pictures were acquired at 10X (background) and 60X (foreground) magnification respectively. Representative images of at least 4 independent fields of two independent experiments

Fig. 4

CD44, ERα/β and PR in BJ human foreskin fibroblasts. A Immunofluorescent (IF) staining. Nuclei were counterstained in blue (DAPI) and cytoskeleton in green (phalloidin-488), while CD44, ERα and PR in red (Alexa-568). Magnification 10X. B-C Gene expression analysis of ERα/β in MCF7, MDA-MB-231 and BJ fibroblasts. D Gene expression analysis of PR in MCF7, MDA-MB-231 and BJ fibroblasts. Histograms represent fold-change in the gene expression of three independent experiments, while error bars represent ± SEM

Fig. 5

Flow cytometry analysis for the detection of ERα and PR in mobilized-PB, fibroblasts, and iPSCs. A-H: representative images of ERα (left) and PR (right) expression in G-CSF mobilized-PB (gated on CD34 + HSPCs, A, Episomal iPSCs (B), BJ fibroblasts (C), BJ iPSCs (D), 253-G1 iPSCs (E), F3 iPSCs (F), and in MDA-MB231 negative control (G) and MCF7 positive control (H); red = aspecific fluorescence, blue = FITC-labeled target. I-L: Histograms representing either ΔMFI = median fluorescence intensity (I,J) or percentage (K,L) of ERα (I,K) and PR expression (J,L) in G-CSF mobilized-PB (gated on CD34 + HSPCs), Episomal iPSCs, BJ fibroblasts, BJ iPSCs, 253-G1 iPSCs, F3 iPSCs, and in MDA-MB231 negative control and MCF7 positive control. Bars represent the mean ± SEM from at least three independent experiments.*, # p < 0.05, **, ##, §§ p < 0.01, ***, ###, §§§ p < 0.001, ****, ####, §§§§ p < 0.0001; * vs MDA-MB231; # vs MCF7; § MDA-MB231 vs MCF7

Fig. 6

Representative images of morphological changes during mammary-like organoids generation from iPSCs. A iPSCs colony; B 1-d mEBs differentiation; C 10-d mEBs differentiation; D Mammary-like organoids. Pictures were acquired at 4X (background) and 20X (foreground) magnification respectively

Fig. 7

H&E and IHC staining. A) H&E stain; B) PAN-CK; C-D-F) Luminal (CK5/7 + and GATA3 +) and E–G) basal cells markers (CK18 + and TP63 +); H) PR and I) ERα. Representative images of mEBs obtained from iPSCs Episomal (I); mEBs obtained from iPSCs BJ (II); mammary-like organoids (III) differentiation

Fig. 8

Gene expression analysis of luminal (CK5/7 + and GATA3 +) and basal markers (CK18 + and TP63 +) markers, PR, ERα and ERβ in iPSCs, 10d-mEBs and 20-d mammary-like organoids. Histograms represent fold-change in the gene expression, while error bars represent ± SEM. *p < 0.05, **p < 0.01