Breastmilk is a novel source of stem cells with multilineage differentiation potential

Abstract

The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration.

Figure 1

Human breastmilk cells from the lactating mammary epithelium express embryonic stem cell (ESC) genes. (A): Immunostaining of normal human resting and lactating breast tissues for expression of ESC genes (OCT4, SOX2, NANOG, SSEA4, and TRA-1-60/81). The absence or minimal expression of ESC genes in the resting breast epithelium is contrasted by the presence of positively stained cells in the alveolar and ductal epithelium of the lactating breast. Positive cells were also observed in the alveolar/ductal lumen, suggesting their presence in breastmilk. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars = 10 μm. (B): Ex vivo immunostaining of freshly isolated breastmilk cells for expression of ESC genes. The first three micrographs show negative control cells (human fibroblasts; scale bars = 50 μm). Nuclei were stained with Hoechst 33342 (blue). Scale bars = 2.5 μm. (C): Fluorescence-activated cell sorting (FACS) analysis of freshly isolated breastmilk cells for expression of ESC genes. The first panel shows negative control cells (human fibroblasts). For each marker, examples of FACS breastmilk cell expression profiles from three participants are shown. (D): Western blotting showing expression of OCT4, SOX2, and NANOG by freshly isolated breastmilk cells. β-Actin was used as a housekeeping gene. -Fibrob.: human fibroblasts (negative control); OTBCs: OTBC-L1 (positive control); S1–S4 breastmilk samples. Abbreviations: APC, allophycocyanin; OTBC, OCT4-transduced breast cells; FITC, fluorescein isothiocyanate.

Figure 2

Breastmilk stem cells expressing ESC genes display clonogeneicity and ESC-like colony morphology and phenotype. (A): Quantitative real-time polymerase chain reaction (PCR) assay for expression of OCT4, SOX2, NANOG, and KLF4 by human breastmilk cells in 17 breastmilk samples (S1–S17), and comparison with human fibroblasts, mammary epithelial cells isolated from resting breast mammoplasties, and hESCs. Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level of the fibroblasts. Bars represent the mean ± SEM. (B): Coculture of human breastmilk-derived stem cells (hBSCs) with mouse embryonic feeder fibroblasts in hESC medium resulted in formation of two distinct types of adherent colonies: ESC-like breastmilk-derived colonies (second panel) with morphology similar to hESCs (cell line H7, first panel); and non-ESC-like breastmilk-derived colonies (third panel) with various morphologies. All ESC-like hBSC colonies expressed OCT4, SOX2, NANOG, SSEA4, SSEA3, and TRA-1-60, similar to hESC line H7. Most non-ESC-like colonies expressed these genes at low levels, if at all. Scale bars = 50 μm. Abbreviation: hESCs, human embryonic stem cells.

Figure 3

Breastmilk stem cells expressing embryonic stem cell (ESC) genes display self-renewal in 3D culture. (A): 3D culture of breastmilk cells and formation of single cell-originated spheroids that expressed ESC genes (OCT4, SOX2, NANOG, SSEA4, and TRA-1-60) and which could be serially passaged, demonstrating self-renewal. Typically, the most rapid increase in spheroid size was observed within the first 1–4 days (first two panels). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars = 20 μm. (B): Time-course of ESC gene expression by breastmilk cells in 3D culture conditions, demonstrating upregulation of ESC genes during the first 12 days of spheroid formation. Quantitative real-time polymerase chain reaction (PCR) assay for expression of OCT4, SOX2, and NANOG in four breastmilk-derived cell samples (S1–S4) from four different participants at day zero (D0 – fresh breastmilk cells prior to culture), and then days 1–12 (D1–D12) of spheroid growth. Each point on the graphs represents the mean ± SEM. Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level of human fibroblasts. For comparison purposes, expression levels of the hESC line H7 are shown in bars (mean ± SEM). More examples are shown in Supporting Information Figure S5.

Figure 4

Breastmilk stem cells differentiate into cells of the three germ layers. (A): Immunostaining for mammoectodermal, neuroectodermal, endodermal, and mesodermal differentiation under corresponding growth conditions. Mammary differentiation: Myoepithelial cells (CK14⁺, SMA⁺) and luminal cells (CK18⁺/CD49f⁻), some of which synthesized milk proteins (β-casein). Neural differentiation: Neuronal-like colonies expressing β-III-tubulin. Hepatocyte differentiation: Initial upregulation of the endodermal progenitor marker OV6 and subsequent upregulation of the mature hepatocyte markers AFP and M2PK, and the functional hepatocyte marker albumin. Pancreatic differentiation: Pancreatic beta cells (PDX1⁺/insulin⁺). Osteogenic differentiation: Colonies with the osteoblastic profile (RUNX2⁺/OSX⁺). Chondrogenic differentiation: Colonies with the chondrocyte marker profile (RUNX2⁺/SOX6⁺). Adipogenic differentiation: PPAR-γ⁺ cells that formed large lipid droplets (oil red O⁺). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars = 20 μm. (B): Western Blot analysis of secreted milk proteins (α-lactalbumin and lactoferrin) in culture supernatants under mammary differentiation conditions. Breastmilk and purified proteins were used as positive controls, and fresh medium as negative control. (C): Quantitative real-time polymerase chain reaction (PCR) assay for insulin expression in breastmilk-derived cultures under pancreatic differentiation conditions. Insulin mRNA expression levels in freshly isolated breastmilk cells (BM-fresh) and breastmilk cells cultured under pancreatic conditions (BM-panc) are compared with those of mammary epithelial cells isolated from resting breast mammoplasties (RB) and human fibroblasts (FB) cultured under the same pancreatic differentiation conditions. Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level of RB. Bars represent the mean ± SEM. Abbreviation: AFP, α-fetoprotein.

Figure 5

Proposed multilineage potential of breastmilk stem cells. Upregulation of the transcription factors (TFs) OCT4, SOX2, and NANOG during pregnancy and lactation results in broader differentiation potential of a mammary stem cell population under specific microenvironmental cues. Accessed via breastmilk, these cells can differentiate in corresponding microenvironments into not only the two main mammary epithelial lineages (green) but also into mesodermal lineages (blue), endodermal lineages (beige), and the neuroectodermal lineage (purple).