Rare Somatic Cells from Human Breast Tissue Exhibit Extensive Lineage Plasticity
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Rare somatic cells from human breast tissue exhibit extensive lineage plasticity Somdutta Roya,b, Philippe Gascarda,b, Nancy Dumonta,b, Jianxin Zhaoa,b, Deng Pana,b, Sarah Petriea,b, Marta Margetab, and Thea D. Tlstya,b,1 aHelen Diller Comprehensive Cancer Center, and bDepartment of Pathology, University of California, San Francisco, CA 94143-0511 Edited* by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved January 18, 2013 (received for review October 31, 2012) We identified cell surface markers associated with repression of us to search for cell surface markers that would offer the op- p16INK4a/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical de- portunity for positive selection, i.e., markers which would exhibit INK4a terminant in the acquisition of a plastic state. These cell surface differential expression in cells with p16 activity compared INK4a INK4a markers allowed direct isolation of rare cells from healthy human with cells with repressed p16 . We observed that p16 breast tissue that exhibit extensive lineage plasticity. This subpopu- repression is relieved upon differentiation into luminal and A lation is poised to transcribe plasticity markers, OCT3/4, SOX2, and myoepithelial cells (Fig. S1 ) and used these cell populations for comparison. Most markers, such as IGF1R (insulin-like growth NANOG, at levels similar to those measured in human embryonic EGFR stem cells and to acquire a plastic state sensitive to environmental factor 1 receptor), (epidermal growth factor receptor), cadherins and integrins, failed to demonstrate differential ex- programming. In vitro, in vivo, and teratoma assays demonstrated pression in the presence or absence of p16INK4a activity (Fig. that either a directly sorted (uncultured) or a single-cell (clonogenic) S1B). CD133, a gene reported to be expressed in neural and cell population from primary tissue can differentiate into functional hematopoietic stem/progenitor cells, was significantly down- derivatives of each germ layer, ectodermal, endodermal, and meso- regulated and thus could not be used for positive selection (11, dermal. In contrast to other cells that express OCT3/4, SOX2, and 12). Strikingly, this analysis identified a dramatic coincident NANOG, these human endogenous plastic somatic cells are mortal, overexpression and down-regulation of CD73 and CD90, re- express low telomerase activity, expand for an extensive but finite spectively (Fig. S1B). To confirm the causal role of p16INK4a in number of population doublings, and maintain a diploid karyotype modulating these two ideal candidates, human mammary epi- CELL BIOLOGY before arresting in G1. thelial cells with low expression of CD73 and high expression of INK4a CD90 were transfected with sh p16 (short hairpin to INK4a NT5E/CD73 | pluripotency p16 ) and assayed for changes in CD73 and CD90 expression INK4a (Fig. S1 C and D). Introduction of sh p16 caused a 77% p16INK4a C urrent observations are expanding our classic perceptions reduction in basal expression (Fig. S1 ). The down-reg- about phenotypic plasticity, i.e., the ability to be shaped or ulation of p16INK4a activity was accompanied by a dramatic in- C crease (>100-fold on average) in the number of cells expressing programmed to a specific cell type. Recent studies indicate that + − CD73 and repressing CD90 (CD73 CD90 cells; Fig. S1D). intestine and skin not only contain classic tissue-specific stem cells + − Having identified CD73 CD90 as a signature for cells with that maintain tissue homeostasis but also independent cell pop- p16INK4a ulations that generate the same range of differentiated cells in repression and a potential for plasticity, we analyzed 10 disease-free human breast tissues (reduction mammoplasties) for response to wound healing (1, 2). We have also learned that + − stromal cues not only dictate which cell population will respond to the presence of CD73 CD90 cells. All tissues were devoid of fi visible disease, bacterial, fungal, or viral contamination, and speci c environmental signals but also which phenotypes will SI Methods manifest. For example, placement of neural stem cells within the exhibited a normal diploid 46, XX karyotype ( and Fig. S2A). Freshly isolated single-cell populations were first de- mammary stroma results in mammary cell differentiation (3). + Thus, cell lineage plasticity can be directed by chemical and me- pleted of the lineage-positive (Lin ) fraction (hematopoietic, endothelial, and fibroblast cells) (Fig. S2B) then fractionated using chanical stromal signals to generate tissues (4). − INK4a the gating shown in Fig. S2C. The resultant lineage-negative (Lin ) Extensive studies have demonstrated that repression of p16 / + population was partitioned into four subpopulations, CD73 CDKN2A (cyclin-dependent kinase inhibitor 2A) is associated with − + + − − CD90 (R1) (5.3%), CD73 CD90 (R2) (1.9%), CD73 CD90 the acquisition of a plastic state, i.e., the ability of a cell to change − + D phenotypes. In epithelial cells, repression of p16INK4a does not only (R3) (84.6%), and CD73 CD90 (R4) (8.2%) (Fig. S2 and inactivate cell cycle arrest in response to stress but also allows in- Dataset S1). creased expression of chromatin remodeling proteins that are im- – portant for epigenetic plasticity underlying differentiation (5). In Differentiation into Three Germ Lineages. Analysis of the R1 R4 Drosophila, the up-regulation of such chromatin remodeling subpopulations by quantitative RT-PCR (qPCR) array revealed distinctive expression of genes in R1 previously reported to proteins sets the expression pattern of pluripotent cells (6). In A – undifferentiated murine myoblasts, an increase in chromatin confer multi- and pluripotency (Fig. 1 ). In contrast to R2 R4, remodeling proteins inhibits differentiation and dictates the R1 expressed several (but not all) of these genes at levels com- parable to human embryonic stem cells (hESCs). Thus, we ex- decision between progenitor and differentiated states (7). Fur- – thermore, mice engineered for knock-out of B-lymphoma Mo- amined the capacity of R1 R4 to differentiate into ectodermal, MLV insertion region 1 homolog (BMI-1), a polycomb protein endodermal, and mesodermal lineages. INK4a that inhibits p16 transcription fail to repress p16INK4a ac- tivity and fail to generate hematopoietic and neural stem cells (8–10). Given these observations, we reasoned that repression of Author contributions: S.R., P.G., and T.D.T. designed research; S.R., N.D., J.Z., D.P., and S.P. p16INK4a might also modulate expression of cell surface markers performed research; S.R., P.G., M.M., and T.D.T. analyzed data; and S.R., P.G., and T.D.T. that could be used for the prospective isolation of cells with wrote the paper. extensive lineage plasticity. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. Results Freely available online through the PNAS open access option. INK4a Selected Cell Surface Markers Regulated by p16 . Comparative 1To whom correspondence should be addressed. E-mail: [email protected]. massively parallel RNA sequencing of human mammary epi- INK4a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thelial cells with or without naturally repressed p16 allowed 1073/pnas.1218682110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218682110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 CD49f (α-6-integrin) and MUC-1 (mucin 1), respectively (Fig. S3B) (16, 17). Flow cytometry identified R1 as the only pop- ulation with multilineage potential (Fig. 1D). The first mam- mosphere passage generated colonies representative of all three mammary lineages. With subsequent passages, the bipotent + + progenitors (CD49f /MUC-1 ) increased over passages 1–3: 20%, 32%, and 66%, respectively, at the expense of the differ- entiated progeny. Using the complementary morphologic col- ony-producing assay, R1 mammosphere-derived (single) cells differentiated into three types of colonies: colonies containing exclusively myoepithelial cells (Fig. S3B, R1, Top), exclusively luminal epithelial cells (Fig. S3B, R1, Middle) or bipotent cells (coexpressing both lineage markers) along with cells of both lineages (Fig. S3B, R1, Bottom). This differentiation potential was maintained in subsequent passages (Fig. 1D). In contrast, R2 and R3 mammosphere-derived cells differentiated pre- dominantly into myoepithelial and luminal colonies or only into luminal colonies, respectively (Fig. 1D and Fig. S3B). In a second assay, acquisition of functional alveolar differen- tiation was assessed by cellular production of human β-casein with human-specific antibodies (Fig. S2E and Fig. 1E). Cells derived from R1–R3 mammospheres were allowed to differentiate on a collagen substratum, overlaid with reconstituted basement membrane gel devoid of growth factors (Matrigel), supplemented with prolactin. Under these conditions, only R1 mammosphere- derived cells produced β-casein, consistent with alveolar differ- Fig. 1. Assessment of R1 for ectodermal multilineage potential. (A) Expres- sion of multi- and pluripotency-associated genes in directly sorted R1–R4 entiation. R2 and R3 produced luminal cells that failed to dif- subpopulations (n = 4) and hESC H7 (n = 3) assayed by qPCR array. Results are ferentiate in the presence of prolactin. We speculate that these expressed as fold changes compared with hESC H9 (n = 3) and normalized to cells are much like the mature luminal cells reported by Lim et al. the housekeeping gene GAPDH.