A co-culture system for studies of paracrine effects of stromal cells on the growth of epithelial cells

Kanazawa, Takuya; Hosick, Howard L.

doi:10.1007/bf01404745

Cited by 5 publications

(5 citation statements)

References 6 publications

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“…Mouse mammary epithelial cells in primary culture responded to salivary mesenchyme cells and mammary fat pad precursor cells of fetal mice, with increased growth rates in a collagen gel matrix and with formation of colonies from single cells in a soft agarose gel. These stimulatory effects on mammary epithelial cells were exerted by diffusible factors released from stromal cells separated from the epithelial target cells by a permeable agarose barrier (Kanazawa and Hosick, 1992). Conditioned medium mimicked the effects of co-cultured stromal cells.…”

Section: Discussionmentioning

confidence: 97%

“…We have not investigated why this difference exists, but we suspect that it relates to the use of collagen gels inside the insert; collagen may block the pores, or alternatively pores may not become fluid filled, thus preventing diffusion of solubilized molecules. The co-culture configuration as used here (Kanazawa and Hosick, 1992) may be preferable when target cells are suspended within a gel. We have also utilized a co-culture system similar to that described by Montesano et al (1991)) in which both cell types are suspended in collagen gels separated by a cell-free collagen gel layer.…”

Section: Discussionmentioning

confidence: 99%

“…Collagen gel co-culture Mesenchyme and epithelium were co-cultured according to the procedure described by Kanazawa and Hosick (1992). Briefly, primary fetal tissues or passaged stromal cells were plated in wells (inside diameter, 16 mm) of 24-well multiwell culture plates and were maintained for 1-2 days in 1 ml of DMEM supplemented with 10% BCS and insulin (5 kgiml).…”

Section: Materials and Methods Animalsmentioning

confidence: 99%

“…Mammary epithelial cells were co-cultured with several different mesenchyme tissues to evaluate whether our culture system could be used to duplicate the different growth responses observed in epithelium in response to these mesenchymes in vivo (Sakakura et al, 1979). A culture medium was prepared that was able t o maintain both cell types but which itself induced only minimal growth of any of the cell types used in this study when they were cultured alone in monolayers or in collagen gels (Kanazawa and Hosick, 1992).…”

Section: Stimulation Of Anchorage-dependent Growth Of Mammary Epithelmentioning

confidence: 99%

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Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes

Kanazawa

Hosick

1992

Journal Cellular Physiology

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When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Materials and Methods Animalsmentioning

confidence: 99%

Section: Stimulation Of Anchorage-dependent Growth Of Mammary Epithelmentioning

confidence: 99%

See 2 more Smart Citations

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes

Kanazawa

Hosick

1992

Journal Cellular Physiology

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“…Cell interactions can occur directly through cell–cell contact and indirectly through the diffusion of factors secreted by one cell that act on the specific receptors of another cell [Grellier et al, ]. To assess these interactions, co‐culture models in which two or more distinct cell populations are grown in the same environment have been used [Kanazawa and Hosick, ]. This approach allows for a partial mimicking of the in vivo environment during in vitro evaluations, keeping co‐cultured cells in direct contact with secreted cytokines and autocrine and paracrine factors [Malekshah et al, ; Bigdeli et al, ].…”

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confidence: 99%

Mesenchymal Stem Cells Repress Osteoblast Differentiation Under Osteogenic‐Inducing Conditions

Santos

Abuna

Castro‐Raucci

et al. 2015

J of Cellular Biochemistry

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This study was designed to investigate the influence of mesenchymal stem cells (MSCs) on osteoblast (OB) differentiation. Rat bone marrow MSCs were cultured either in growth medium that maintained a MSC phenotype or in osteogenic medium that induced differentiation into OBs. Then, cells were grown in two different culture conditions: indirect co-culture of MSCs and OBs and OBs cultured in MSC-conditioned medium. As a control culture condition, OBs were grown in osteogenic medium without the influence of MSCs. We evaluated cell proliferation, the gene expression of key bone markers, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) expression, and extracellular matrix mineralization. The results showed that, regardless of whether OBs were indirectly co-cultured with MSCs or cultured in MSC-conditioned medium, MSCs repressed OB differentiation, as evidenced by the downregulation of all evaluated bone marker genes, decreased ALP activity, inhibition of BSP protein expression, and reduced extracellular matrix mineralization. Taken together, these results indicate that despite the key role of both MSCs and OBs in the osteogenic process, the repressive effect of MSCs on OB differentiation in an osteogenic environment may represent a barrier to the strategy of using them together in cell-based therapies to induce bone repair.

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Three-dimensional mammary primary culture model systems

Darcy

1996

J Mammary Gland Biol Neoplasia

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Model systems have been developed to investigate the complex and coordinated regulation of mammary gland development and transformation. Primary cultures, using newly isolated cells or tissue, are optimal for such studies since, in comparison to immortalized cell lines, the normal signal transduction pathways are presumed to be intact. Three such models are described, including whole organ culture, mammary epithelial cell (MEC) organoids, and MEC-stromal cocultures. Studies using whole-organ culture have the advantage that the normal glandular architecture remains intact, the MEC can undergo lobuloalveolar development and express milk proteins in a hormone dependent manner, and, following hormonal withdrawal, undergo involution. Moreover, transformation of the MEC is readily accomplished. Culture of isolated MEC organoids within an EHS-derived reconstituted basement membrane permits extensive proliferation, branching end bud and alveolar morphogenesis, and accumulation of milk protein and lipid in a physiologically relevant hormone- and growth factor-dependent manner. This model can thus be utilized to investigate the mechanism by which various modulators exert their direct effects on the epithelium. Finally, in view of compelling evidence for stromal-epithelial interactions during normal mammary gland development, and potentially also during the development of malignancy, models in which MEC can be cocultured with enriched populations of stroma offer considerable potential as a tool to understand the nature and mechanisms of the interactions that occur during the various developmental states, and how such interactions may go awry during carcinogenesis.

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A co-culture system for studies of paracrine effects of stromal cells on the growth of epithelial cells

Cited by 5 publications

References 6 publications

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes

Mesenchymal Stem Cells Repress Osteoblast Differentiation Under Osteogenic‐Inducing Conditions

Three-dimensional mammary primary culture model systems

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