title: Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
abstract: BACKGROUND
  Cancer 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.
  
  
  METHODS
  Single 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.
  
  
  RESULTS
  We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) 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 beta-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.
  
  
  CONCLUSIONS
  Thus, 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.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
authors.forename: Devaveena
authors.initials: D
authors.lastname: Dey
authors.forename: Meera
authors.initials: M
authors.lastname: Saxena
authors.forename: Anurag N
authors.initials: AN
authors.lastname: Paranjape
authors.forename: Visalakshi
authors.initials: V
authors.lastname: Krishnan
authors.forename: Rajashekhar
authors.initials: R
authors.lastname: Giraddi
authors.forename: M Vijaya
authors.initials: MV
authors.lastname: Kumar
authors.forename: Geetashree
authors.initials: G
authors.lastname: Mukherjee
authors.forename: Annapoorni
authors.initials: A
authors.lastname: Rangarajan
mesh_terms: D000818:Animals; D051927:CD24 Antigen / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D042361:Mammary Glands, Human / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008954:Models, Biological; D014411:Neoplastic Stem Cells / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D051927:CD24 Antigen