Adipose-derived mesenchymal stem cells formed acinar-like structure when stimulated with breast epithelial cells in three-dimensional culture

Abstract

Lipotransfer has been applied in breast augmentation surgery for several years and the resident adipose-derived stem cells (ASCs) play an important role in enhancing fat graft survival. However, the interaction between ASCs and mammary epithelium is not fully understood. Many studies have shown that ASCs have a tumor-supportive effect in breast cancer. To the best of our knowledge, this is the first study on the effect of mammary epithelial cells on the human ASCs in 3D culture. ASCs were cultivated on matrigel in the conditioned medium (CM) prepared from a human breast epithelial cell line (HBL-100). The ASCs formed KRT18-positive acini-like structures after stimulation with breast epithelial cells. The expression of epithelial genes (CDH1 and KRT18) was up-regulated while the expression of mesenchymal specific genes (CDH2 and VIM) was down-regulated as determined by qRT-PCR. The stemness marker (CD29) and angiogenic factors (CD31 and VEGF) were also down-regulated as examined by immunofluorescence. In addition, the CM obtained from HBL-100 enhanced the migration and inhibited the adipogenic differentiation of ASCs. These results demonstrate that ASCs have the ability to transform into epithelial-like cells when cultured with mammary epithelial cells. Given these observations, we infer that ASCs have a positive effect on lipotransfer, not only due to their ability to secrete growth factors, but also due to their direct participation in the formation of new breast tissue.

Fig 1

Multi-differentiation (adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation) of human adipose derived stem cells (ASCs) and surface markers of ASCs assessed by flow cytometry A: Morphological observation of ASCs under an inverted light microscope. B: Alizarin Red staining of osteogenic differentiation on 21st day. C: Oil Red O staining of adipogenic differentiation on 21st day. D: Toliudine Blue staining of chondrogenic differentiation. E: Positive and negative surface markers of ASCs determined by flow cytometry: ASCs expressed CD105, CD73 and CD90, and lack expression of CD45, CD34, and CD14. Scale bar = 100μm (A-D).

Fig 2. Changes of ASCs in 3D culture.

A: CCK8 assay. The y-axis shows the amount of absorbance, the y-axis the measuring time points. B: The Transwell migration assay results. The y-axis shows the cells number per mm2 and the x-axis shows the different groups. * P<0.05, ***P<0.001, n = 3. Scale bar = 40μm. C: ASCs and HASCs were underwent 21 days of adipogenic differentiation. The cells were fixed and stained with Oil-Red-O. The images were took by a microscope. Scale bar = 50μm. D: Quantitative analysis of Oil Red O staining. Dye stained cells was extracted with isopropanol and the absorbance at 540 nm was measured. * P<0.05, ***P<0.001, n = 3.

Fig 3. Different behaviors of ASCs induced by HBL-100 conditioned medium in 3D culture.

A: The Day 0 referred to the day in which ASCs had undergone serum starvation. In the control group (ASC), the branching structure formed by ASCs continued growing on day 5 and formed a network-like structure on day 10. In the conditional medium group (HASCs), the branching structure formed an acinar-like structure on day 5 which failed to grow larger on day 10. B: Cell contraction. C: Cell assembly. D: Spinal Cell Contraction. Scale bar = 40μm (A-C).

Fig 4. KRT18 expression and mRNA expression of ASCs induced by HBL-100 conditioned medium in 3D culture.

A: The relative expression of epithelial specific genes CDH1, KRT18 was significantly higher in HASCs compared with ASCs while the relative expression of mesenchymal specific genes CDH2, VIM was significantly lower. MRNA expression was measured by quantitative RT-PCR. 2^-△△ CT method was used to calculate relative expression of genes. * * P<0.01, *** P<0.001, n = 3. B: KRT18 (green) expression in 3D culture. Nuclei were stained with DAPI (blue). C: Human mammary tissue was observed with HE staining. KRT18-specific antibody was used to mark human mammary epithelial cells or epithelial-like cells. Scale bar = 40μm (B and C).

Fig 5. Analyses of stem cell markers and angiogenic factors expression by immunofluorescence showing CM downregulated the stemness and angiogenic differentiation of ASCs in 3D.

Representative fluorescence images of ASCs and HASCs stained for KRT8, stem cell markers (CD29, CD90) and angiogenic factors (CD31, VEGF). Nuclei were stained with DAPI (blue). Scale bar = 20μm.