Accumulation of Multipotent Progenitors with a Basal Differentiation Bias during Aging of Human Mammary Epithelia

Garbe, James C.; Pépin, François; Pelissier, Fanny A.; Sputova, Klara; Fridriksdottir, Agla J.; Guo, Diana E.; Villadsen, René; Park, Morag; Petersen, Ole W.; Borowsky, Alexander D.; Stampfer, Martha R.; LaBarge, Mark A.

doi:10.1158/0008-5472.can-12-0157

Cited by 101 publications

(173 citation statements)

References 48 publications

(62 reference statements)

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“…To do so, we used uncultured primary cells isolated from human reduction mammoplasty tissue. Compared with the fourth-passage primary cells used in our previous experiments, uncultured primary cells have elevated levels of cellular heterogeneity in a variety of cell-surface markers (15,27). Consistent with underlying variability in the energetics of their cellular interfaces, we found that pure populations of uncultured luminal or myoepithelial cells formed aggregates with more variable and overlapping circularity than fourth-passage primary cells (Fig.…”

Section: Self-organization Of the Mammary Gland Is Robust To Highly Vsupporting

confidence: 85%

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

Cerchiari

Garbe

Jee³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Developing tissues contain motile populations of cells that can selforganize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its selforganization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo-and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.heterogeneity | cell sorting | differential adhesion | mammary | prostate S elf-organization is a process that contributes to pattern formation and repair at all scales of biological complexity. At the tissue scale, defining robust strategies of self-organization is critical for engineering functional tissues, as well as for understanding development and the breakdown of tissue structure during diseases such as cancer (1). During development, two or more populations of motile cells can self-organize into spatially ordered tissues by a process referred to as cell sorting (2-4). The outcome of cell sorting can be rationalized using physical models that invoke cell-type-specific differences in interfacial energies. Interfacial energies arise through the action of a contractile cell cortex coupled to adhesion molecules (e.g., cadherins) that link the cortices of neighboring cells and signal to modulate cortical tension at specific cellular interfaces (5). In general, the organization of a tissue after cell sorting corresponds to a configuration that maximizes the formation of the most energetically favorable (hereafter referred to as most cell-cell cohesive) † cellular interfaces (6). For example, with an intermediate level of heterotypic cell-cell cohesion the most self-cohesive cell type is typically found in the tissue core, with the le...

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Section: Self-organization Of the Mammary Gland Is Robust To Highly Vsupporting

confidence: 85%

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

Cerchiari

Garbe

Jee³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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107

120

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“…[45][46][47] Our M87A/85 media support proliferation of pre-stasis HMEC with progenitor lineage markers, and allow robust proliferation prior to p16 upregulation. 10,48 Such lower stress/p16-inducing conditions may be reflective of early stage carcinogenesis in vivo, if unstressed progenitor cells are initial targets.…”

Section: Discussionmentioning

confidence: 99%

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

et al. 2014

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Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient nonclonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16INK4 ; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional "passenger" errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of "passenger" genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.

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“…Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation [4][5][6][7][8][9][10][11][12][13][14][15]16 . The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic 5,15 .…”

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confidence: 99%

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

LaBarge¹,

Garbe²,

Stampfer³

2013

JoVE

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Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells [1][2] . Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells 3 . The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using lowstress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor) 4-5

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Accumulation of Multipotent Progenitors with a Basal Differentiation Bias during Aging of Human Mammary Epithelia

Cited by 101 publications

References 48 publications

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

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