Generation of Mosaic Mammary Organoids by Differential Trypsinization

Rubio, Stefany; Cazares, Oscar; Macias, Hector; Hinck, Lindsay

doi:10.3791/60742

Cited by 6 publications

(11 citation statements)

References 22 publications

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“…To determine whether ROBO1 promotes alveolar differentiation through its action specifically in one of these cell types, we generated organoids that are mosaic for Robo1 expression ( Fig. 4 A) ( Rubio et al, 2020 ). Robo1−/− basal cells were mixed with Robo1+/+ luminal cells (KO/WT) and, vice versa, Robo1+/+ basal cells were mixed with Robo1−/− luminal cells (WT/KO) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Primary cell organoids were grown as previously described ( Rubio et al, 2020 ). Briefly, primary cells were mixed and grown in Matrigel Growth Factor Reduced (GFR), Phenol Red-Free (Corning, CB-40230C) and cultured in basal medium.…”

Section: Methodsmentioning

confidence: 99%

“…To induce differentiation, primed HC11 cells were cultured in DIP Medium [RPMI-1640, supplemented with 10% FBS, 5 µg/ml insulin, 1 µM dexamethasone, 1× anti-anti and 3 µg/ml Prolactin (NHPP, oPRL-21)] at 37°C with 5% CO 2 . Primary cells were harvested from 8- to 12-week-old mice and cultured as previously described ( Macias et al, 2011 ; Rubio et al, 2020 ).…”

Section: Methodsmentioning

confidence: 99%

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Alveolar progenitor differentiation and lactation depends on paracrine inhibition of Notch via ROBO1/CTNNB1/JAG1

et al. 2021

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In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating β-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Alveolar progenitor differentiation and lactation depends on paracrine inhibition of Notch via ROBO1/CTNNB1/JAG1

et al. 2021

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“…Fibroblast attachment to substrates is comparatively less robust as opposed to hepatocytes and we exploited this aspect to separate the two cell populations based on differential trypsinization time. The process of differential trypsinization to separate fibroblasts from other cells have been used widely over several years due to the faster trypsinization of fibroblast [ 55 , 56 , 57 , 58 , 59 ].…”

Section: Discussionmentioning

confidence: 99%

“…Cultures were thoroughly observed under a microscope to ensure that only primary hepatocytes remained attached to the substrates following the first trypsinization step. The process of differential trypsinization to separate fibroblasts from other cells have been used widely over several years due to the faster trypsinization of fibroblast [ 55 , 56 , 57 , 58 , 59 ]. To qualitatively validate the selective trypsinization technique, we replated the trypsinized cell suspension onto collagen-coated TCPS and observed that the cell attached were primarily fibroblasts.…”

Section: Methodsmentioning

confidence: 99%

Exploring Interactions between Primary Hepatocytes and Non-Parenchymal Cells on Physiological and Pathological Liver Stiffness

Natarajan

Moeun

Kidambi

2021

Biology

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Chronic liver disease is characterized by progressive hepatic fibrosis leading to the formation of cirrhosis irrespective of the etiology with no effective treatment currently available. Liver stiffness (LS) is currently the best clinical predictor of this fibrosis progression irrespective of the etiology. LS and hepatocytes-nonparenchymal cells (NPC) interactions are two variables known to be important in regulating hepatic function during liver fibrosis, but little is known about the interplay of these cues. Here, we use polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to study how cell–cell interaction and stiffness regulates hepatocytes function. Specifically, primary rat hepatocytes were cocultured with NIH-3T3 fibroblasts on soft (2 kPa) and stiff substrates that recreates physiologic (2 kPa) and cirrhotic liver stiffness (55 kPa). Urea synthesis by primary hepatocytes depended on the presence of fibroblast and was independent of the substrate stiffness. However, albumin synthesis and Cytochrome P450 enzyme activity increased in hepatocytes on soft substrates and when in coculture with a fibroblast. Western blot analysis of hepatic markers, E-cadherin, confirmed that hepatocytes on soft substrates in coculture promoted better maintenance of the hepatic phenotype. These findings indicate the role of stiffness in regulating the hepatocytes interactions with NPCs necessary for maintenance of hepatocytes function.

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Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

Molinuevo,

Menendez,

Cadle

et al. 2024

Nat Commun

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Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation required for lactation is unknown. Here, we show that DNA damage accumulates due to replication stress during pregnancy, activating the DNA damage response. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents reveals that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. We identify a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with homozygous loss of Wee1 resulting in decreased endoreplication, alveologenesis and milk production. Thus, we propose that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. Our study sheds light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.

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Generation of Mosaic Mammary Organoids by Differential Trypsinization

Cited by 6 publications

References 22 publications

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of Notch via ROBO1/CTNNB1/JAG1

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of Notch via ROBO1/CTNNB1/JAG1

Exploring Interactions between Primary Hepatocytes and Non-Parenchymal Cells on Physiological and Pathological Liver Stiffness

Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

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