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. STEM Cells2012;30:2164-2174.

OBJECTIVE: To observe the effect of electroacupuncture (EA) and acupuncture (A) on the proliferation of stem cells in the subependymal zone (SPZ) of the lateral ventricle and the frontal lobe cortex in hyperlipemia(HL) combined with cerebral ischemia (CI) rats. METHODS: A total of 72 male SD rats were randomized into control, HL, HL+EA, CI, CI+A, HL+CI, HL+CI+EA I and HL+CI+EA II groups (n=9 /group). HL model was established by feeding the animals with high fat forage for 6 weeks and CI model was established by FeCl3-induced occlusion of the unilateral middle cerebral artery. EA was applied to "Sanyinjiao" (SP 6) and "Fenglong" (ST 40) once daily for 17 days for HL+ EA group; and acupuncture to "Baihui" (GV 20) and "Shuigou" (GV 26) once daily for 7 days for CI + A group. For HL+CI+EA I group, EA was applied to SP 6 + ST 40 first before CI, once daily for 10 days, followed by EA of SP 6+ST 40 and acupuncture of GV20+GV26 for 7 days after CI. For HL+CI+EA II group, no treatment was given before CI, then, acupuncture of GV 20 + GV 26 and EA of SP 6 + ST 40 were given once daily for 7 days after CI. The immunoactivity of Nestin and proliferation cell nuclear antigen (PONA) of SPZ was detected by immunohistochemistry. RESULTS: In comparison with normal control group, the numbers of both Nestin and PCNA immunoreaction (IR) positive cells in the dorsolateral extension and the wall of the lateral ventricle of the brain increased significantly in CI and HL+CI groups (P<0.01). Compared with CI group, the numbers of Nestin and PCNA IR positive cells in the dorsolateral extension and the wall of the lateral ventricle in CI + A group increased considerably (P<0.01). In comparison with HL+CI group, both Nestin and PCNA IR positive cell numbers in the dorsolateral extension and the wall of the lateral ventricle of the brain in HL+CI+EA I and HL+CI+EA II groups increased significantly (P<0.01), and the effect of HL+CI+EA I group was markedly superior to that of HL+CI+EA II group in upregulating the numbers of Nestin and PCNA IR positive cells in the aforementioned regions of the lateral ventricle (P<0.01). No significant differences were found between HL and control groups, and between HL+EA and HL groups in the numbers of Nestin and PCNA IR positive cells in the dorsolateral extension and the wall of the lateral ventricle of the brain (P>0.05). CONCLUSION: EA can upregulate Nestin and PCNA expression of the dorsolateral extension and the wall of the lateral ventricle of the brain on the ischemic side in rats with CI, and with HL+CI, which may contribute to its effects in promoting the proliferation and migration of neural stem cells in the brain.

Stem cells in mammary tissue have been well characterised by using the mammary stem cell marker, cytokeratin (CK) 5 and the mature epithelial markers CK14, CK18 and CK19. As these markers have never been reported in cells from breastmilk, the aim of this study has been to determine whether mammary stem cells are present in expressed human breastmilk. Cultured cells from human breastmilk were studied by using immunofluorescent labelling and reverse transcription/polymerase chain reaction (RT-PCR). We found a heterogeneous population of cells with differential expression of CK5, CK14, CK18 and CK19. Further, by using the multipotent stem cell marker, nestin, we identified cells in culture that were positive only for nestin or double-positive for CK5/nestin, whereas no co-staining was observed for CK14, CK18 and CK19 with nestin. When cells isolated from breastmilk were analysed by using RT-PCR prior to culture, only nestin and CK18 were detected, thereby indicating that breastmilk contained differentiated epithelial and putative stem cells. Furthermore, fluorescence-activated cell-sorting analysis demonstrated, in breastmilk, a small side-population of cells that excluded Hoechst 33342 (a key property of multipotent stem cells). When stained for nestin, the cells in the side-population were positive, whereas those not in the side-population were negative. The presence of nestin-positive putative mammary stem cells suggests that human breastmilk is a readily available and non-invasive source of putative mammary stem cells that may be useful for research into both mammary gland biology and more general stem cell biology.