Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

<h4>Background</h4>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 su...

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Autores principales:	Devaveena Dey, Meera Saxena, Anurag N Paranjape, Visalakshi Krishnan, Rajashekhar Giraddi, M Vijaya Kumar, Geetashree Mukherjee, Annapoorni Rangarajan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/153aa133d40b45b1aca4062391a0cf55
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spelling	oai:doaj.org-article:153aa133d40b45b1aca4062391a0cf552021-11-25T06:23:03ZPhenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.1932-620310.1371/journal.pone.0005329https://doaj.org/article/153aa133d40b45b1aca4062391a0cf552009-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19390630/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology</h4>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.<h4>Principal findings</h4>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.<h4>Conclusions</h4>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.Devaveena DeyMeera SaxenaAnurag N ParanjapeVisalakshi KrishnanRajashekhar GiraddiM Vijaya KumarGeetashree MukherjeeAnnapoorni RangarajanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 4, p e5329 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Devaveena Dey Meera Saxena Anurag N Paranjape Visalakshi Krishnan Rajashekhar Giraddi M Vijaya Kumar Geetashree Mukherjee Annapoorni Rangarajan Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
description	<h4>Background</h4>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.<h4>Methodology</h4>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.<h4>Principal findings</h4>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.<h4>Conclusions</h4>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.
format	article
author	Devaveena Dey Meera Saxena Anurag N Paranjape Visalakshi Krishnan Rajashekhar Giraddi M Vijaya Kumar Geetashree Mukherjee Annapoorni Rangarajan
author_facet	Devaveena Dey Meera Saxena Anurag N Paranjape Visalakshi Krishnan Rajashekhar Giraddi M Vijaya Kumar Geetashree Mukherjee Annapoorni Rangarajan
author_sort	Devaveena Dey
title	Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
title_short	Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
title_full	Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
title_fullStr	Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
title_full_unstemmed	Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
title_sort	phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/153aa133d40b45b1aca4062391a0cf55
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Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

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