- Email: [email protected]
P28. Primary mammary epithelial cells from the lactating epithelium have distinct growth and differentiation capacity
S26
Abstract / Differentiation 80 (2010) S17–S63
relatively high the follicles did not mature to the antral stages. Two oocytes containing follicles were frequently observed in Wnt4 knock-out ovaries. The estrous-circle was also disturbed, the diestrus was prolonged and fertility was reduced by 63% in comparison to wild-type mice. The qRT-PCR showed decreased expression of KitL where as expression of Bmp15, Gdf9 and Nobox was not changed significantly. We also fate mapped the cell lineages generated from the cells that express Wnt-4 gene normally by using a GFPCre knock in allele to the Wnt-4 locus. These studies revealed the Wnt-4 expressive cells generated most of the follicular cells. The kidneys of the Wnt-4 deficient mice were compromised. As the Amhr2Cre recombines a floxed reporter also in the kidney of an adult we draw a conclusion that Wnt-4 has a signaling role in the adult kidney as well. The hypomorphic Wnt-4 mCherry knock-in allele demonstrated severe follicular defects that lead to reduced fertility. We conclude that Wnt-4 signaling maintains female fertility by supporting maturation of the follicle and is essential for proper kidney function in the adult.
Using such models may be useful to further understand mechanisms that underlie mammary gland pathologies. doi: 10.1016/j.diff.2010.09.034
P29 Regulation of TGF-b1 in intercellular junctions in epithelial ovarian carcinomas
Angela Barone, Yihong Zhu, Jingfang Gao, Karin Sundfeldt Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Sweden E-mail address: [email protected] (J. Gao)
doi: 10.1016/j.diff.2010.09.033
P28 Primary mammary epithelial cells from the lactating epithelium have distinct growth and differentiation capacity
E. Thomas a, N. Zeps a,b, P. Rigby b, P. Hartmann a a
The University of Western Australia, Perth, Australia St. John of God Pathology, Perth, Australia E-mail address: [email protected] (E. Thomas) b
The mammary gland is a unique organ in its ability to undergo repetitive cycles of growth, differentiation, function and involution in the mature adult. These processes depend upon a pool of committed multipotent cells that undergo asymmetric division to give rise to more differentiated progeny that proliferate at the onset of gestation. It is normally not possible to access these cells from lactating women to generate primary mammary epithelial cultures for research as surgical intervention is rare during lactation. We have identified cells expressing markers for multipotent mammary epithelial cells (CD49f, CD24 and p63) in human breastmilk samples. These primary mammary epithelial cells (PMEC) can be expanded and manipulated in epithelial-specific monolayer culture to produce differentiated cells expressing luminal (CK18) and myoepithelial (CK14) markers. Using the immortalised non-tumorigenic mammary line MCF10A and tumorigenic mammary line MDA231 as a comparison, we show that PMEC have a distinctive response to extra-cellular matrix (ECM) and growth and functional regulators. In monolayer culture PMEC bypass replicative senescence at day 8 in response to ethanolamine and hormones prolactin, placental lactogen and RANKL, while immortal lines show no response in their rapid growth pattern. In three-dimensional ECM culture PMEC and MCF10A produce alveolus-like structures that maintain a basal layer of CD49f-positive cells and a luminal layer of CK18-positive cells, while MDA231 form disorganized invasive colonies. Uniquely, PMEC also produce hallmark milk proteins beta-casein and alpha-lactalbumin in response to prolactin stimulation in the luminal secretory layer. These data imply that the cell population expanded during lactation is receptive to distinct stimuli that results in differentiation in ways not observed in cells taken from the non-lactating gland. We demonstrate that human milk is a non-invasive source of these cells, providing a tool for further research into the specific pathways that regulate differentiation.
Epithelial ovarian cancer (EOC) commonly thought to arise from ovarian surface epithelia (OSE) covering the ovaries is the fifth most common cause of cancer related death in females in the Western world. It is the most lethal form of all gynecological malignancy due to the diagnosis at late stages. The pathogenesis of EOC and mechanisms underlying this event remain poorly defined. Epithelial–mesenchymal transition (EMT), a process with loss of polarity and disruption of intercellular junctions, is vital for embryonic morphogenesis as well as cancer metastasis. We have shown that TGF-b1 can induce EMT in OSE in vitro. In this study, we investigated whether TGF-b1 induces EMT in the cultured ovarian cancer cell line, OVCAR-3. We studied phenotype and trans-epithelial resistance in a filter culture insert system. Regulation of TGF-b1 in cell–cell adhesion was examined by analyzing the expression of tight junction proteins, occludin, claudin-1, -3, -4 and -7, and adherens junction proteins Ncadherin and E-cadherin using Western blotting and their mRNA expression by qPCR. We did not observe any cell phenotypic difference between TGF-b1 treated and untreated OVCAR-3. However, we did find a decrease of trans-epithelial resistance in TGF-b1 treated cells concomitant with downregulated protein and/or RNA expressions of E-cadherin, occludin as well as claudin-3, and -4, -7 whereas upregulated N-cadherin and claudin 1 in TGF-b1 treated cells. Our results suggest that TGF-b1 can modulate the formation of tight junction and adherens junction and the ion-barrier function of tight junction in OVCAR-3 without transition to a mesenchymal phenotype. doi: 10.1016/j.diff.2010.09.035
P30 Compartmental microfluidic culture system of embryonic stem cell-derived neurons
Byung Man Lee, Seul Ki Min, Sung Hoon Kim, Chung Hwan Shin, Hwa Sung Shin Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea E-mail address: [email protected] (H.S. Shin)
A neuron has axon and somata which show distinctly different functions. For this reason, compartmental culture of neurons has been studied as a useful research tool for axonal pathology. Primary neurons, which have been cultured in compartmental microfluidic devices, are limited to be prepared in time and location. In contrast,
Abstract / Differentiation 80 (2010) S17–S63
relatively high the follicles did not mature to the antral stages. Two oocytes containing follicles were frequently observed in Wnt4 knock-out ovaries. The estrous-circle was also disturbed, the diestrus was prolonged and fertility was reduced by 63% in comparison to wild-type mice. The qRT-PCR showed decreased expression of KitL where as expression of Bmp15, Gdf9 and Nobox was not changed significantly. We also fate mapped the cell lineages generated from the cells that express Wnt-4 gene normally by using a GFPCre knock in allele to the Wnt-4 locus. These studies revealed the Wnt-4 expressive cells generated most of the follicular cells. The kidneys of the Wnt-4 deficient mice were compromised. As the Amhr2Cre recombines a floxed reporter also in the kidney of an adult we draw a conclusion that Wnt-4 has a signaling role in the adult kidney as well. The hypomorphic Wnt-4 mCherry knock-in allele demonstrated severe follicular defects that lead to reduced fertility. We conclude that Wnt-4 signaling maintains female fertility by supporting maturation of the follicle and is essential for proper kidney function in the adult.
Using such models may be useful to further understand mechanisms that underlie mammary gland pathologies. doi: 10.1016/j.diff.2010.09.034
P29 Regulation of TGF-b1 in intercellular junctions in epithelial ovarian carcinomas
Angela Barone, Yihong Zhu, Jingfang Gao, Karin Sundfeldt Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Sweden E-mail address: [email protected] (J. Gao)
doi: 10.1016/j.diff.2010.09.033
P28 Primary mammary epithelial cells from the lactating epithelium have distinct growth and differentiation capacity
E. Thomas a, N. Zeps a,b, P. Rigby b, P. Hartmann a a
The University of Western Australia, Perth, Australia St. John of God Pathology, Perth, Australia E-mail address: [email protected] (E. Thomas) b
The mammary gland is a unique organ in its ability to undergo repetitive cycles of growth, differentiation, function and involution in the mature adult. These processes depend upon a pool of committed multipotent cells that undergo asymmetric division to give rise to more differentiated progeny that proliferate at the onset of gestation. It is normally not possible to access these cells from lactating women to generate primary mammary epithelial cultures for research as surgical intervention is rare during lactation. We have identified cells expressing markers for multipotent mammary epithelial cells (CD49f, CD24 and p63) in human breastmilk samples. These primary mammary epithelial cells (PMEC) can be expanded and manipulated in epithelial-specific monolayer culture to produce differentiated cells expressing luminal (CK18) and myoepithelial (CK14) markers. Using the immortalised non-tumorigenic mammary line MCF10A and tumorigenic mammary line MDA231 as a comparison, we show that PMEC have a distinctive response to extra-cellular matrix (ECM) and growth and functional regulators. In monolayer culture PMEC bypass replicative senescence at day 8 in response to ethanolamine and hormones prolactin, placental lactogen and RANKL, while immortal lines show no response in their rapid growth pattern. In three-dimensional ECM culture PMEC and MCF10A produce alveolus-like structures that maintain a basal layer of CD49f-positive cells and a luminal layer of CK18-positive cells, while MDA231 form disorganized invasive colonies. Uniquely, PMEC also produce hallmark milk proteins beta-casein and alpha-lactalbumin in response to prolactin stimulation in the luminal secretory layer. These data imply that the cell population expanded during lactation is receptive to distinct stimuli that results in differentiation in ways not observed in cells taken from the non-lactating gland. We demonstrate that human milk is a non-invasive source of these cells, providing a tool for further research into the specific pathways that regulate differentiation.
Epithelial ovarian cancer (EOC) commonly thought to arise from ovarian surface epithelia (OSE) covering the ovaries is the fifth most common cause of cancer related death in females in the Western world. It is the most lethal form of all gynecological malignancy due to the diagnosis at late stages. The pathogenesis of EOC and mechanisms underlying this event remain poorly defined. Epithelial–mesenchymal transition (EMT), a process with loss of polarity and disruption of intercellular junctions, is vital for embryonic morphogenesis as well as cancer metastasis. We have shown that TGF-b1 can induce EMT in OSE in vitro. In this study, we investigated whether TGF-b1 induces EMT in the cultured ovarian cancer cell line, OVCAR-3. We studied phenotype and trans-epithelial resistance in a filter culture insert system. Regulation of TGF-b1 in cell–cell adhesion was examined by analyzing the expression of tight junction proteins, occludin, claudin-1, -3, -4 and -7, and adherens junction proteins Ncadherin and E-cadherin using Western blotting and their mRNA expression by qPCR. We did not observe any cell phenotypic difference between TGF-b1 treated and untreated OVCAR-3. However, we did find a decrease of trans-epithelial resistance in TGF-b1 treated cells concomitant with downregulated protein and/or RNA expressions of E-cadherin, occludin as well as claudin-3, and -4, -7 whereas upregulated N-cadherin and claudin 1 in TGF-b1 treated cells. Our results suggest that TGF-b1 can modulate the formation of tight junction and adherens junction and the ion-barrier function of tight junction in OVCAR-3 without transition to a mesenchymal phenotype. doi: 10.1016/j.diff.2010.09.035
P30 Compartmental microfluidic culture system of embryonic stem cell-derived neurons
Byung Man Lee, Seul Ki Min, Sung Hoon Kim, Chung Hwan Shin, Hwa Sung Shin Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea E-mail address: [email protected] (H.S. Shin)
A neuron has axon and somata which show distinctly different functions. For this reason, compartmental culture of neurons has been studied as a useful research tool for axonal pathology. Primary neurons, which have been cultured in compartmental microfluidic devices, are limited to be prepared in time and location. In contrast,