The HC11 cell line was kindly provided by Dr. D. Medina (Houston, TX). The authors are grateful to Dr. Andrew Craig of Queen&#39;s University for many reagents and valuable suggestions. EpH4 cells were a gift from Dr. C. Roskelley (UBC, Vancouver). Colleen Schick provided excellent technical assistance for 3D culture studies.
The financial assistance of the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Canadian Breast Cancer Research Alliance, the Ontario Centres of Excellence, the Breast Cancer Action Kingston (BCAK) and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. BE received grant support from CIHR, CBCF, BCAK and Cancer Research Society Inc. PTG is supported by a Canada Research Chair, Canadian Foundation for Innovation, CIHR, NSERC and Canadian Cancer Society. MN was supported by a studentship from the Terry Fox Training Program in Transdisciplinary Cancer Research from NCIC, a Graduate award (QGA), and a Dean&#39;s Award from Queen&#39;s University. MG was supported by postdoctoral fellowships from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#39;s University. VH was supported by a CBCF doctoral fellowship and a postdoctoral fellowship from the Terry Fox Foundation Training Program in Transdisciplinary Cancer Research in partnership with CIHR. Hanad Adan was the recipient of an NSERC summer studentship. BS was supported by a Queen&#39;s University graduate award.

1. Plating HC11 Cells
<ol>
	<li>In a laminar flow hood using sterile techniques prepare a bottle with 50 mL of HC11 cell medium: RPMI-1640 with 10% fetal bovine serum (FBS), 5 &#956;g/mL insulin, and 10 ng/mL EGF (see Table of Materials).</li>
	<li>Plate approximately 400,000 cells per 3 cm Petri dish: Pass two 10 cm, 50% confluent Petri dishes into twenty 3 cm dishes in HC11 medium. The cells must be well spread out but drying must be avoided.
	<ol>
		<li>Aspirate the medium into a flask using a vacuum pump.</li>
		<li>Add 250 &#181;L of trypsin (see Table of Materials) per 10 cm plate. Swirl and hit the plate from the sides while keeping it horizontal to spread the trypsin and to dislodge the attached cells</li>
		<li>Observe under phase contrast microscopy with a 4x objective to make sure the cells have started to detach from the edges before pipetting.</li>
		<li>Aspirate approximately 1.5 mL of HC11 medium in a sterile, 9 inch Pasteur pipette and squirt it vertically against the cells. Rotate the Petri dish while squirting to dislodge all cells. You must work fast to avoid drying of the cells.</li>
		<li>Transfer all cells to the bottle with the HC11 medium and swirl.</li>
		<li>Pipette 2 mL of cell suspension in each 3 cm Petri dish. Rock the Petri dishes crosswise to spread evenly. Place the cells in a 37 &#176;C, 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>The next day, or when cells are ~90&#8211;100% confluent, aspirate the medium, and replace it with medium with FBS and insulin but lacking EGF.</li>
</ol>
2. Differentiation Induction, Monitoring, and Quantitation of HC11 Cells
<ol>
	<li>After growing the cells in medium lacking EGF for 24 h, add the differentiation medium (HIP medium) to ten 3 cm plates: RPMI-1640 supplemented with 10% FBS, 1 &#956;g/mL Hydrocortisone (see Table of Materials), 5 &#956;g/mL Insulin and 5 &#956;g/mL Prolactin (see Table of Materials) for up to 10 days.</li>
	<li>Keep the other ten 3 cm plates as controls: Change to a medium with 10% FBS and 5 &#956;g/mL insulin lacking EGF and HIP.</li>
	<li>Change the medium every 2&#8211;3 days to both HIP-treated cells and controls. Pipette the medium carefully to make sure that cells do not detach.</li>
	<li>To monitor differentiation (i.e., the formation of &#34;domes&#34;), observe the cells under phase contrast microscopy (Figure 1A, left panel). If the cells are expressing green fluorescence protein (GFP), observe under fluorescence microscopy (excitation = 485/20; emission = 530/25; magnification 240x; 20x objective) (Figure 1A, right panel).</li>
	<li>To quantitate the degree of differentiation, extract proteins from one Petri dish each of HIP-treated cells and controls, once a day for a total of 10 days and probe Western blots for &#946;-casein (see Table of Materials) (Figure 1B).
	<ol>
		<li>Scrape the cells on ice with 1.5 mL ice-cold phosphate-buffered saline (PBS) into a 1.8 ml plastic centrifuge tube.</li>
		<li>Pellet the cells at 350 x g, 1 min, 4 &#176;C.</li>
		<li>Quickly wash the pellet 2x with ice-cold PBS. Drain any residue.</li>
		<li>Make a lysis buffer: 50 mM HEPES (pH = 7.4), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 1% NP-40, 100 mM NaF, 2 mM Na3VO4, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), 10 &#956;g/mL aprotinin, 10 &#956;g/mL leupeptin16. Add 100 &#181;L per 3 cm Petri dish.</li>
		<li>Clarify the lysate by centrifugation at 10,000 x g for 10 min in the cold.</li>
		<li>Determine protein concentration (see Table of Materials) and load 10&#8211;20 &#181;g of protein from each sample onto a 10% polyacrylamide-SDS gel.</li>
		<li>Electrophorese for 15 h at 45 V, or at 100 V for ~2&#8211;2.5 h.</li>
		<li>Transfer onto a nitrocellulose or PVDF membrane using an electroblotting transfer apparatus (see Table of Materials).</li>
		<li>Block with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST).</li>
		<li>Add the primary antibody, goat anti-&#946;-casein diluted 1:2,000 or mouse anti-&#946; actin diluted 1:1,000 (see Table of Materials).</li>
		<li>Wash 3x with TBST, 5 min each time.</li>
		<li>Add the secondary antibody, horseradish peroxidase (HRP) linked donkey anti-goat diluted 1:2,500, or HRP linked horse anti-mouse antibody diluted 1:10,000.</li>
		<li>Wash 3x with TBST, 5 min each time.</li>
		<li>Add ECL reagents to the membrane according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
</ol>
3. Plating and 3D Growth of EpH4 Cells in EHS Matrix
NOTE: The matrix is liquid at temperatures &#60;10 &#176;C and solid at temperatures above. Store at -80 &#176;C. Thaw at 4 &#176;C the night before use. Pre-chill the tissue culture plates and pipette tips at -20 &#176;C prior to handling and keep the matrix, plates, and pipette tips on ice to prevent it from solidifying.
<ol>
	<li>Grow EpH4 cells in 2D in RPMI-1640 medium with 10% FBS and 5 &#181;g/mL insulin (EGF is not required).</li>
	<li>Prepare EpH4 growth medium supplemented with 10% (v/v, 3,500 &#181;L) or 20% (v/v 2,000 &#181;L) matrix in two 50 mL conical tubes. Keep on ice until ready to use.</li>
	<li>Coat 10 wells (1 cm2 each) of a 24 well plate with 150 &#956;L of undiluted matrix. Spread the matrix using a pipette tip. Avoid creating bubbles. Gently tap the sides of the plate while holding it horizontally to ensure that the matrix is evenly distributed across the bottom of the well.</li>
	<li>Incubate the 24 well plate at 37 &#176;C for 1 h to allow the matrix to solidify.</li>
	<li>Trypsinize EpH4 cells as described above for HC11 cells and count them with a hemocytometer. Transfer 5 x 104 cells per well to a 1.5 mL sterile conical centrifuge tube and spin at 250 x g for 5 min.</li>
	<li>Carefully aspirate the medium and place the tube on ice.</li>
	<li>Resuspend cells in 350 &#181;L of EpH4 growth medium supplemented with 20% matrix using a 1 mL pipette tip while keeping the conical tube on ice. Avoid bubbles. It is important to ensure a single cell suspension.</li>
	<li>Once the bottom layer matrix has solidified (the matrix should appear translucent and lighter in color compared to the initial coating of the wells), add the 350 &#181;L of cell suspension to each coated well and place in a CO2 incubator at 37 &#176;C for ~1 h to allow the 20% matrix layer to solidify.
	<ol>
		<li>Observe the cells microscopically under phase contrast with a 4x objective. Single cells should be visible.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of EpH4 medium containing 10% matrix on top of the 350 &#181;L of cells suspended in 20% matrix. Incubate at 37 &#176;C.</li>
</ol>
4. Differentiation Induction of EpH4 Cells Grown in 3D (Figure 2)
NOTE: Besides differentiation, EpH4 cells can also undergo tubulogenesis when stimulated with HGF (hepatocyte growth factor) in 3D culture. Tubular outgrowths can be seen after 10 days of HIP and HGF stimulation.
<ol>
	<li>Begin differentiation induction of EpH4 cells 1 day after plating in the matrix. Carefully remove 150 &#181;L of top medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 &#181;L of EpH4 medium containing HIP and 10% matrix.</li>
	<li>Replace the 10% matrix-HIP medium every 2 days for up to 10 days. Grow control cells in the same 10% matrix medium without HIP.</li>
	<li>Monitor mammosphere formation under phase contrast (Figure 3A, lower panel).</li>
</ol>
5. Tubulogenesis Induction of EpH4 Cells Grown in 3D (Figure 3A and 3C)
<ol>
	<li>Prepare 24 well plates and EpH4 cells for growth in matrix as detailed in steps 3.1&#8211;3.9. The day after plating the cells in the matrix, remove 150 &#181;L of EpH4 medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 &#181;L of EpH4 medium containing 10% matrix, HIP, and 20 ng/mL HGF (see Table of Materials).</li>
	<li>Replace the 10% matrix/HIP/HGF medium every 2 days for up to 12&#8211;14 days.</li>
	<li>Monitor tubule formation microscopically using a 20x or 40x objective (Figure 3A, right panel).</li>
</ol>
6. Quantitation of Differentiation: Western Blotting for &#946;-casein
<ol>
	<li>Carefully pipette off the 10% matrix-HIP medium from the wells. Rinse the 20% matrix layer 2x with 350 &#181;L of ice-cold PBS. The spheroids should still be present in the matrix layer.</li>
	<li>Add 700&#8211;1,000 &#181;L of ice-cold PBS with 1 mM ethylenediaminetetraacetic acid (EDTA) directly into the wells. Gently detach the bottom layer of the 100% matrix from the well with a pipette tip to recover spheroids present in that layer. Shake gently for 30 min at 4 &#176;C.</li>
	<li>Carefully transfer the spheroid suspension to a conical tube. Rinse the wells with ~500 &#181;L of PBS-EDTA to recover any remaining spheroids in the wells and add to the conical tube.</li>
	<li>Rock on ice for an additional 30 min. Make sure the matrix has fully dissolved. If visible matrix clumps are seen, add more PBS-EDTA or shake longer.</li>
	<li>Centrifuge the solution to pellet the spheroids at 350 x g for 5 min.</li>
	<li>Aspirate the supernatant, lyse the spheroids in ice-cold lysis buffer, and probe for &#946;-casein, cyclin D1, and p120RasGAP by Western blotting as in step 2.5 above16. As primary antibodies, use goat anti-&#946;-casein diluted 1:2,000, rabbit anti-cyclin D1 diluted 1:2,000, and mouse anti-p120 diluted 1:2,000. For secondary antibodies, use HRP linked donkey anti-goat diluted 1:2,500, HRP linked donkey anti-rabbit diluted 1:2,500, and HRP linked horse anti-mouse diluted 1:10,000.</li>
</ol>

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The authors have no conflicts to disclose.

HC11 cells are ideally suited for the study of differentiation in conjunction with neoplastic transformation. An added advantage is that HC11 cells are easily infectable with Mo-MLV-based retroviral vectors to express a variety of genes. In our hands, EpH4 cells were more difficult to infect with the same retroviral vectors than HC112.
Cell-to-cell contact and growth arrest are key prerequisites for HC11 cell differentiation. Thus, to achieve uniform differentiation acr...

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors in a three-dimensional organization, and this is mimicked in culture by high density cell growth. Cell-to-cell adhesion is mediated mainly through cadherin receptors, which define cell and tissue architecture. Interestingly, it was recently demonstrated that cadherins also play a powerful role in signal transduction, especially in survival signaling1. Paradoxically, some of these cell-to-cell adhesion signals emanating from cadherins were recently found to be shared by both differentiation and neoplasia2. Here, we describe methods of induction and assessment of differentiation in two representative types of mouse breast epithelial cell lines, HC11 and EpH4.
The HC11 mouse breast epithelial cell line can provide a useful model for the study of epithelial cell differentiation. HC11 cells are a COMMA-1D-derived cell line, originating from the mammary gland of a mid-pregnant Balb/c mouse3. In contrast to other COMMA-1D derivative clones, the HC11 clone has no requirement for exogenously added extracellular matrix or cocultivation with other cell types for the in vitro induction of the endogenous &#946;-casein gene by lactogenic hormones3. This cell line has been used extensively in differentiation studies because it has retained important characteristics of the normal mammary epithelium: HC11 cells can partially reconstitute the ductal epithelium in a cleared mammary fat pad4. Moreover, they can differentiate in a two-dimensional (2D) culture when grown to confluence attached to a plastic Petri dish surface in the presence of a steroid such as Hydrocortisone or Dexamethasone, in addition to Insulin and Prolactin (HIP medium) lacking epidermal growth factor (EGF), an inhibitor of differentiation5,6,7. Under these conditions, HC11 cells produce milk proteins such as &#946;-casein and WAP, which are detectable by Western blotting within 4 days following induction. At the same time, a portion of HC11 cells forms rudimentary mammary gland-like structures termed &#34;domes&#34; in a stochastic manner. Domes are visible 4&#8211;5 days following induction and gradually increase in size up to day 10, concomitant with an increase in &#946;-casein production8. Interestingly, HC11 cells possess mutant p539, and therefore represent a preneoplastic state. For this reason, the HC11 model is ideally suited to study signaling networks of differentiation in conjunction with neoplasia in the same cell system.
EpH4 cells, a derivative of IM-2 cells, are a nontumorigenic cell line originally derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a mid-pregnant Balb/c mouse10. EpH4 cells form continuous epithelial monolayers in 2D culture, but do not differentiate into glandular-like structures10,11. However, following 3D growth in a material consisting of a mixture of extracellular matrix proteins produced by EHS mouse sarcoma cells12 (EHS matrix, matrix, or Matrigel, see Table of Materials), in addition to stimulation with HIP, EpH4 cells can recapitulate the initial stages of mammary gland differentiation. Under these conditions, EpH4 cells form spheroids (also called mammospheres) that exhibit apical-basal polarity and a hollow lumen, and are capable of producing the milk proteins &#946;-casein and WAP, similar to lactating mammary epithelial cells. Contrary to HC11 cells, which are undifferentiated, and some express mesenchymal markers13, EpH4 cells exhibit a purely luminal morphology14. EpH4 cells have also been reported to produce milk proteins in 2D culture through stimulation with dexamethasone, insulin, and prolactin15. However, this approach precludes the study of regulatory effects that mimic the mammary gland microenvironment in 3D culture.

<table><tbody><tr><td>30% Acrylamide/0.8% Bis Solution</td><td>Bio Rad</td><td>1610154</td><td>Western Blotting</td></tr><tr><td>Anti-Cyclin D1 antibody</td><td>rabbit, Santa Cruz</td><td>sc-717</td><td>Western Blotting</td></tr><tr><td>Anti-p120 antibody</td><td>mouse, Santa Cruz</td><td>sc-373751</td><td>Western Blotting</td></tr><tr><td>Anti-&amp;beta; actin antibody</td><td>mouse, Cell Signalling technology</td><td>3700</td><td>Western Blotting</td></tr><tr><td>Anti-&amp;beta; casein antibody</td><td>goat, Santa Cruz Biotechnology</td><td>sc-17971</td><td>Western Blotting</td></tr><tr><td>Aprotinin</td><td>Bio Shop</td><td>APR600</td><td>Lysis Buffer</td></tr><tr><td>Bicinchoninic Acid Solution</td><td>Sigma</td><td>B9643-1L-KC</td><td>Protein Determination</td></tr><tr><td>Bovine serum albumin</td><td>Bio Shop</td><td>ALB007.500</td><td>Protein Determination</td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio Rad</td><td>170-5061</td><td>Western Blotting</td></tr><tr><td>Copper(II) sulphate</td><td>Sigma</td><td>C2284-25ML</td><td>Protein Determination</td></tr><tr><td>DAPI</td><td>Thermofisher Scientific</td><td>D1306</td><td>Staining</td></tr><tr><td>Digitonin</td><td>Calbiochem, Cedarlane Laboratories Ltd</td><td>14952-500</td><td>Staining</td></tr><tr><td>EDTA</td><td>Bio Shop</td><td>EDT001.500</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>Sigma</td><td>E9644</td><td>Medium for HC11 Cells</td></tr><tr><td>Fetal calf serum</td><td>PAA</td><td>A15-751</td><td>Cell Culture Medium</td></tr><tr><td>Goat- Anti Rabbit-HRP</td><td>Santa Cruz</td><td>SC-2004</td><td>Western Blotting</td></tr><tr><td>Hepatocyte Growth Factor recombinant</td><td>Gibco</td><td>PHG0321</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>7365-45-9</td><td>Cell Culture</td></tr><tr><td>Horse Anti-Mouse HRP</td><td>Cell Signalling Technology</td><td>7076</td><td>Western Blotting</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888</td><td>HIP Medium</td></tr><tr><td>insulin</td><td>Sigma</td><td>I6634</td><td>HIP Medium</td></tr><tr><td>Laminar-flow hood</td><td>BioGard Hood</td><td></td><td>Cell Culture</td></tr><tr><td>Leupeptin</td><td>Bio Shop</td><td>LEU001.10</td><td>Lysis Buffer</td></tr><tr><td>Matrix (Engelbreth-Holm-Swarm matrix, Matrigel)</td><td>Corning</td><td>CACB 354230</td><td></td></tr><tr><td>Mouse Anti-Goat HRP</td><td>Santa Cruz</td><td>sc-8360</td><td>Western Blotting</td></tr><tr><td>Mowiol 4-88 Reagent</td><td>Calbiochem, Cedarlane Laboratories Ltd</td><td>475904-100GM</td><td>Staining</td></tr><tr><td>Multi-photon confocal microscope</td><td>Leica TCS SP2</td><td></td><td></td></tr><tr><td>Na3VO4</td><td>Bio Shop</td><td>SOV850</td><td>Lysis Buffer</td></tr><tr><td>Na4P207</td><td>Sigma</td><td>125F-0262</td><td>Lysis Buffer</td></tr><tr><td>NaCl</td><td>Bio Shop</td><td>SOD001.1</td><td>Western Blotting</td></tr><tr><td>NaF</td><td>Fisher Scientific</td><td>7681-49-4</td><td>Lysis Buffer</td></tr><tr><td>Nikon digital Camera</td><td>Coolpix 995</td><td></td><td></td></tr><tr><td>Nitrocellulose</td><td>Bio Rad</td><td>1620112</td><td>Western Blotting</td></tr><tr><td>NP-40</td><td>Sigma</td><td>9016-45-9</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>30525-89-4</td><td>Staining</td></tr><tr><td>Phase Contrast Microscope</td><td>Olympus IX70</td><td></td><td>Cell Culture</td></tr><tr><td>Phenylmethylsuphonyl fluoride</td><td>Sigma</td><td>329-98-6</td><td>Lysis Buffer</td></tr><tr><td>pMX GFP Rac G12V</td><td>Addgene</td><td>14567</td><td></td></tr><tr><td>Prolactin</td><td>Sigma</td><td>L6520</td><td>HIP Medium</td></tr><tr><td>RPMI-1640</td><td>Sigma</td><td>R8758</td><td>Cell Culture Medium</td></tr><tr><td>Tissue Culture Dish 35</td><td>Sarstedt</td><td>83.3900.</td><td>Cell Culture</td></tr><tr><td>Tissue Culture Plate-24 well</td><td>Sarstedt</td><td>83.1836.300</td><td>Cell Culture</td></tr><tr><td>Transfer Apparatus</td><td>CBS Scientific Co</td><td>EBU-302</td><td>Western Blotting</td></tr><tr><td>Tris Acetate</td><td>Bio Shop</td><td>TRA222.500</td><td>Western Blotting</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>9002-07-7.</td><td>Cell Culture</td></tr><tr><td>Tween-20</td><td>Bio Shop</td><td>TWN510.500</td><td>Western Blotting</td></tr><tr><td>Veritical Gel Electrophoresis System</td><td>CBS Scientific Co</td><td>MGV-202-33</td><td>Western Blotting</td></tr></tbody></table>

differentiation of mouse breast epithelial hc11 and eph4 cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

It has long been known that the differentiation of epithelial cells and adipocytes requires confluence and engagement of cadherins2. We and others demonstrated that cell-to-cell adhesion and engagement of E- or N-cadherin and cadherin-11, as occurs with the confluence of cultured cells, triggers a dramatic increase in the activity of the small GTPases Rac and cell division control protein 42 (Cdc42), and this process leads to activation of interleukin&#8211;6 (IL6) family cytokines and Stat3 (sign...

Watch this Scientific Journal Video about Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells at JoVE.com

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

differentiation-of-mouse-breast-epithelial-hc11-and-eph4-cells

Research

JoVE Journal

Cancer Research

8.8K Views.  Queen's University, Kingston. De HC11- en EpH4-cellen zijn epitheelcellijnen van muizenborsten die zich kunnen onderscheiden in de cultuur. Tegelijkertijd kunnen deze cellen worden getransformeerd door een aantal oncogenen. Deze cellen zijn ideaal voor de studie van de onderlinge relatie tussen differentiatie en de neoplastische transformatie.Deze technieken kunnen worden gebruikt in signaaltransductiestudies om doelen voor kankertherapie te ontdekken. Bijvoorbeeld, de kleine GTPase Rac en andere moleculen kunnen l...

Video: Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Developmental Biology

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

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 cells1-2. 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 cells3. 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 low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
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 transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Biology

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioengineering

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Neuroscience

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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