Additional Supporting Information may be found in the online version of this article.

sc-12-0539_sm_SupplFigure1.tif99KSupplemental Figure S1. Ex vivo FACS analysis of ESC gene expression by freshly isolated breastmilk cells (n=12). (a) Gating strategy used to analyse FACS protein expression by breastmilk cells. A first gate was applied in the Forward versus Side Scatter plot as shown. In this gate, only the nucleated cells were selected via PI staining. The gated nucleated cell population was then separated into dead and viable cells, and only the latter were analysed for marker expression. (b) Distribution of the standardised differences in mean fluorescence intensity relative to controls (SDT-C) for ESC genes.
sc-12-0539_sm_SupplFigure2.tif165KSupplemental Figure S2. Ex vivo co-expression of ESC genes by single freshly isolated breastmilk cells. (a) Double immunostaining of single cells demonstrating co-expression of ESC genes. Nuclei were stained with DAPI (blue). Scale bars: 5 μm. (b) FACS analysis of triple-stained cells co-expressing OCT4, SOX2 and NANOG.
sc-12-0539_sm_SupplFigure3.tif53KSupplemental Figure S3. Quantitative real-time PCR assay for expression of additional ESC genes by breastmilk cells and comparison with human fibroblasts and hESCs. Expression of hTERT, REX1 and GDF3 is shown for 12-13 freshly isolated breastmilk cell samples from different participants (S1-S13). For hTERT and REX1, expression of spheroids at day 9 of growth (D9) is also shown, with a marked upregulation of mRNA expression under spheroid conditions. The great variability in mRNA expression between individuals is noted. Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level of the fibroblasts. Bars represent the mean ± SEM.
sc-12-0539_sm_SupplFigure4.tif386KSupplemental Figure S4. Breastmilk-derived cells display ESC-like traits in the absence of MEFs in 2D and 3D growth conditions, in contrast to mammary epithelial cultures derived from mammoplastic reductions of resting breast tissue. (a) Adherent ESC-like colonies of breastmilk cells grown in the absence of MEFs. Phase contrast micrograph of a colony with ESC morphology. Immunostaining of these colonies for OCT4 expression. (b) Mammary epithelial cell cultures derived from mammoplastic reductions of resting breast tissue did not express ESC genes. (c) Various sizes and morphologies of spheroids were obtained from 3D culture of hBSCs. Spheroids in passage 0-2 are shown. (d) Co-expression of ESC genes by single spheroid cells. Nuclei were stained with DAPI (blue). Scale bars: 20 μm.
sc-12-0539_sm_SupplFigure5.tif107KSupplemental Figure S5. Additional examples of time-course of OCT4 expression by breastmilk cells in 3D culture conditions. Quantitative real-time PCR assay for expression of OCT4 in 13 breastmilk-derived cell samples (S1-S13) from 13 participants at day zero (D0 – fresh breastmilk cells prior to culture) and up to day 9 (D9) of spheroid growth. Each sample is identified by a different colour. Fibroblasts and hESCs are shown in black. Each time point was assayed in triplicate or duplicate depending on the number of cells available, and each bar represents the mean ± SEM. Samples S9- S13 are shown only for one time point due to inadequate material to run more time points. Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level of human fibroblasts. Depending on the sample, a variable response of OCT4 expression to 3D culture was seen, with some samples that upregulated OCT4 expression to levels significantly higher than those of hESCs (e.g. S6, S12), and others that either increased OCT4 expression marginally (e.g. S3) or increased it to levels closer to those of hESCs (e.g. S5, S8, S9, S11).
sc-12-0539_sm_SupplFigure6.tif371KSupplemental Figure S6. Spontaneous differentiation of hBSCs into cells from the three germ layers. (a-h) Phase Contrast micrographs of differentiated breastmilk-derived adherent colonies on day 14-21 of culture: (a) epithelial cells; (b) myoepithelial cells; (c) neuron-like cells; (d) fibroblast-like, stromal cells; (e) cobblestone-like cells; (f-h) mixed cell types. (i) Immunostaining of mixed mammary colonies (CK14+/CK18+), stromal cells (Vimentin, STRO-1), myoblast-like cells (desmin, SMA), endodermal colonies (α-fetoprotein - AFP, OV6), and neuron-like cells (β-III-tubulin). Nuclei were stained with DAPI (blue). Scale bars: 50 μm.
sc-12-0539_sm_SupplFigure7.tif176KSupplemental Figure S7. Formation of fat globules within the cells under mammary differentiation conditions of hBSCs, suggesting milk production. (a-c) Phase Contrast micrographs of mammary colonies. Inserts show higher magnifications of cells with lipid droplets. Scale bars: 50 μm.
sc-12-0539_sm_SupplFigure8.tif307KSupplemental Figure S8. Directed differentiation of hBSCs in 3D conditions. (a) Immunostaining of β-casein in mammospheres grown under mammary differentiation conditions. (b) Immunostaining of insulin in pancreatospheres grown under pancreatic differentiation conditions. (c) Immunostaining of cardiac T troponin in cardiospheres grown under cardiomyocyte differentiation conditions. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 μm.
sc-12-0539_sm_SupplTable1.pdf85KSupplemental Table 1.
sc-12-0539_sm_SupplTable2.pdf35KSupplemental Table 2.
sc-12-0539_sm_SupplTable3.pdf37KSupplemental Table 3.
sc-12-0539_sm_SupplTable4.pdf37KSupplemental Table 4.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.