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Preadipocytes stimulate ductal morphogenesis and functional differentiation of human mammary epithelial cells on 3D silk scaffolds - PubMed

Preadipocytes stimulate ductal morphogenesis and functional differentiation of human mammary epithelial cells on 3D silk scaffolds

Xiuli Wang et al. Tissue Eng Part A. 2009 Oct.

Abstract

Epithelial-mesenchymal interactions play an important role in regulating normal tissue development as well as tumor development for the mammary gland, but much is yet to uncover to reach a full understanding of their complexity. To address this issue, the establishment of relevant, surrogate, three-dimensional (3D) human tissue culture models is essential. In the present study, a novel 3D coculture system was developed to study the interactions between human mammary epithelial cells (MCF10A) and adipocytes, a prominent stromal cell type in native breast tissue. The MCF10A cells were cultured within a mixture of Matrigel and collagen in 3D porous silk scaffolds with or without predifferentiated human adipose-derived stem cells (hASCs). The presence of hASCs inhibited MCF10A cell proliferation, induced both alveolar and ductal morphogenesis, and enhanced their functional differentiation as evidenced by histology and functional analysis. The alveolar structures formed by cocultures exhibited proper, immature polarity when compared with native breast tissue. In contrast, only alveolar structures with reverted polarity were observed in the MCF10A monocultures. The effect of ductal morphogenesis in cocultures may correlate to hepatocyte growth factor secreted by the predifferentiated hASCs, based on results from a cytokine blocking assay. Taken together, this in vitro coculture model on silk scaffolds effectively reconstitutes a physiologically relevant 3D microenvironment for epithelial cells and stromal cells and provides a useful system to study tissue organization and epithelial morphogenesis in normal or diseased breast development.

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Figures

FIG. 1.
FIG. 1.

Scanning electron microscopy image of porous silk scaffolds (pore size, 500–650 μm).

FIG. 2.
FIG. 2.

A three-step experimental design for the coculture on silk scaffolds. Stage I, inducing hASCs cells differentiation into predifferentiated hASCs with the differentiation medium I in cell culture flasks; Stage II, maintaining the phenotype of predifferentiated hASCs with the maintenance medium II in cell culture flasks, image showing the cytoplasmic triglyceride droplets in the differentiated hASCs (oil red-O staining); Stage III, coculturing MCF10A cells and predifferentiated hASCs with the medium III on three-dimensional silk scaffolds. hASCs, human adipose–derived stem cells. Color images available online at

www.liebertonline.com/ten

.

FIG. 3.
FIG. 3.

(A) DNA quantification by PicoGreen DNA content assay showed cells proliferation in the monoculture or cocultures models on three-dimensional silk scaffolds. (B) Inhibition effect of predifferentiated hASCs on MCF10A cell proliferation assayed by semi-quantitive analysis of Ki67 staining. Only positive staining cells within epithelial structures were counted in different microscopic fields (n = 10 per slide, 3 slides per group). A significant decrease of the Ki67+ MCF10A cells was observed in the cocultures during the first week, and the percentage of positive staining cells deceased over time (*p < 0.05, #p > 0.05).

FIG. 4.
FIG. 4.

Growth profile and viability of MCF10A cells in the monoculture or coculture constructed on silk scaffold (at day 6). Confocal images showed both alveolar and ductal-like structures formed in cocultures (A), while only alveolar structures were observed in the monocultures (B) (red, MCF10A cells labeled with DiI; green, hASCs labeled with CMFDA). An ideal viability of MCF10A cells in different groups detected by Live/Dead staining (C, D). Hematoxylin and eosin staining showed detailed morphological characteristics of the epithelial structures formed by MCF10A cells in different culture groups (E, F). Clearer lumen structures were observed in the cocultures (F). Arrowhead denotes ductal-like structure; asterisk denotes alveolar structure. Color images available online at

www.liebertonline.com/ten

.

FIG. 5.
FIG. 5.

Immunostaining images showed the morphological characteristics of the epithelial structures formed by cocultured MCF10A cells on silk scaffold. (A) Collagen staining showed the basement membrane formation of the alveolar structure. (B) Positive E-cadherin staining indicated the integrity of the tissue-like structures. (C) Higher casein expression level was observed in cocultures (C1) than in the monocultures (C2). (D) Antibody replaced by PBS served as negative control, and cell nuclei were counterstained by propidium iodide. Color images available online at

www.liebertonline.com/ten

.

FIG. 6.
FIG. 6.

Immunostaining images of sialomucin (red) and GM130 (green) showed the correct polarity of the alveolar structures formed by cocultured MCF10A cells in silk scaffold (B1–B3, day 6). Cell nuclei were counterstained by DAPI (blue), while reversed polarity was observed in the monoculture (C1–C3, day 6). Human breast tissue served as positive control (A1–A3). Color images available online at

www.liebertonline.com/ten

.

FIG. 7.
FIG. 7.

Transcript levels of (α-casein (MCF10A cells, A) and hepatocyte growth factor (hASCs cells, B) by real-time RT-PCR at the indicated time points. Significantly increased expression of casein-α was observed in MCF10A cells cocultured with predifferentiated hASCs (n = 3, *p < 0.05, #p > 0.05).

FIG. 8.
FIG. 8.

Growth profile of MCF10A cells cocultured with predifferentiated hASCs in an eight-well chamber slide after different concentrations of anti-hHGF R (c-met) were supplemented into the coculture medium. A dose-dependent inhibition effect of anti-hHGF R (c-Met) on MCF10A cell ductal morphogenesis was observed as evidenced by the decreased number and shorter length of ductal-like structures in coculture. hHGF, human hepatocyte growth factor.

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