Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Abstract

BackgroundCancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages.MethodologySingle cell suspensions derived from human breast ‘organoids’ were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres.Principal FindingsWe show that primary mammospheres contain a distinct side-population (SP) that displays a CD24low/CD44low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44high/CD24low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated β-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture.ConclusionsThus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

“…In keeping with this, a reverse transcriptase-PCR (RT-PCR) analysis comparing HMECs grown as monolayers versus mammospheres revealed an increased expression of stemness-related genes (such as, Bmi1, Nanog, Lef1, Gli1, Notch1, Hes1, HeyL and CD44), and epithelial to mesenchymal transition (EMT) markers associated with stemness (such as, Vimentin, N-cadherin, FN1, Snai1 and Slug1) (Mani et al, 2008) in mammospheres ( Figure 1a). We had previously demonstrated that the lifespan of normal mammospheres in vitro is limited to four passages (Dey et al, 2009), thereby limiting their utility for studying mammary stem cell biology. Therefore, we sought to generate immortalized breast cell lines that retain long-term stem-like properties.…”

“…We previously reported that normal mammospheres can be passaged in suspension for a maximum of four passages, beyond which they fail to generate spheres and largely undergo senescence (Dey et al, 2009). On the other hand, immortalized mammary cell line such as HMLEs (Elenbaas et al, 2001) can be cultured in vitro for prolonged periods; however, they lack the ability to generate mammospheres (Mani et al, 2008;Morel et al, 2008), indicating the lack of stem-like properties.…”

“…Mammosphere and cell culture Mammospheres were cultured as described previously (Dey et al, 2009). Normal and cancerous breast tissues were obtained from Kidwai Memorial Institute of Oncology (KMIO), Bangalore, as per the Institutional Review Board and in compliance with the ethical guidelines of KMIO and Indian Institute of Science.…”

A Monoclonal Antibody against Human Notch1 Ligand–Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem–like Cells

“…The preliminary evidence also suggests that it can affect the primary breast tumor cells. Inhibition of Notch signaling leads to reduction in mammosphere-forming capacity (19,41,43), hallmark of putative stem-like cell subpopulation. The mAb 602.101 inhibited cell proliferation and mammosphere formation up to 3 generations in cell lines, as well as, the primary tumor cells, suggesting a strong therapeutic prospect of this antibody.…”

“…Mammosphere assay MCF-7, MDA-MB-231 (5 Â 10 4 cells per well), and the enzymatically dissociated single-cell suspensions (2.5 Â 10 5 cells per well) of the primary breast cancer tissues were seeded in a serum-free DMEM-F12 medium with growth factors in a semisolid medium containing methylcellulose as described earlier (19,41). Effect of anti-Notch1 antibodies on sphere-forming efficiency of these cells was assessed by incubating the cells with or without antibodies (10 mg/mL) and culturing for 1 week, replenishing the antibody every fourth day.…”