Adipo-Epithelial Transdifferentiation in In Vitro Models of the Mammary Gland

Abstract

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others' previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and β-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal-epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level.

Keywords: adipocytes; cell culture; cellular transdifferentiation; mammary gland; pregnancy.

Figure 1

qRT-PCR analysis of ELF5 (A), β-casein (B), WAP (C) and SPP1 (D) expression in freshly isolated MEO (T0) and MEO at 7 and 14 days of culture under hormonal stimulation (H). Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01 compared with MEO T0. Data were analyzed using one-way ANOVA.

Figure 2

Phase contrast light microscopy of freshly isolated (T0) MEO embedded in Matrigel (A), MEO grown for 7 days (B) and 14 days (C) in the presence of pregnancy hormones in a serum-free culture medium. Electron Microscopy of MEO without hormones (D); MEO after pregnancy hormones addition (E): alveolar differentiation with lumen and microvilli (arrows); (F): enlargement of an alveolar structure showing hypertrophic Golgi complex (dotted area), glycogen cluster (Gly), and well-developed microvilli (arrow), all signs of alveolar differentiation. Bar: 1 µm in (D) 2 µm in (E) and 0.3 µm in (F).

Figure 3

qRT-PCR analysis of adipogenic markers (AdipoQ and Plin1) in adipocytes co-cultured in absence/presence of T7 (top panel) and T14 (bottom panel) differentiated-MEO and in absence/presence of pregnancy hormones. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 between biological groups as indicated. Data were analyzed using one-way ANOVA.

Figure 4

qRT-PCR analysis of reprogramming markers in adipocytes co-cultured in absence/presence of T7 (top panel) and T14 (bottom panel) differentiated-MEO and in absence/presence of pregnancy hormones. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01 between biological groups as indicated. Data were analyzed using one-way ANOVA.

Figure 5

qRT-PCR analysis of epithelial markers (A) and representative immunoblot and quantification of E-cadherin expression (B) in adipocytes co-cultured in absence/presence of T7 (top panel) and T14 (bottom panel) differentiated-MEO and in absence/presence of pregnancy hormones. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 between biological groups as indicated. Data were analyzed using one-way ANOVA. In B, MEOs treated with hormones for 14 days (MEO+H) were used as positive control.

Figure 6

qRT-PCR (A) analysis of pinking markers and representative immunoblot and quantification of ELF5 expression (B) in adipocytes co-cultured in absence/presence of T7 (top panel) and T14 (bottom panel) differentiated-MEO and in absence/presence of pregnancy hormones. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01 between biological groups as indicated. Data were analyzed using one-way ANOVA. In B, MEO treated with hormones for 14 days (MEO+H) were used as positive control.

Figure 7

qRT-PCR (A) analysis of milk markers and representative immunoblot and quantification of β-casein expression (B) in adipocytes co-cultured in absence/presence of T7 (top panel) and T14 (bottom panel) differentiated-MEO and in absence/presence of pregnancy hormones. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 between biological groups as indicated. Data were analyzed using one-way ANOVA. In (B), MEOs treated with hormones for 14 days (MEO+H) were used as positive control.

Figure 8

Bar plot of mir200c miRNA (x-axis) representing the proportions of counts specific to the considered conditions: A+MEO+H and All other joined conditions (A, A+H, A+MEO, A+Matrix, A+Matrix+H) (y-axis).

Figure 9

qRT-PCR analysis of AdipoQ (A), Plin1 (B) and ELF5 (C), in hMADS adipocytes maintained for 24 h under osteopontin 500 ng/mL (SPP1), pregnancy hormones (H) and H+SPP1. Data (n = 3) are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with untreated hMADS adipocytes (NT). Data were analyzed using one-way ANOVA.