Name: quantification of self-renewal in murine mammosphere cultures
Uploaded: 2019-11-26T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Quantification of Self-renewal in Murine Mammosphere Cultures at JoVE.com

We thank Bruno Amati for the kind gift of the Rosa26-MycER transgenic mouse model. This work was funded by grants from WWCR, AIRC, ERC, and the Italian Ministry of Health to P.G.P. T.V. and X.A. were supported by FIRC and A.S. by a FUV grant.

In vivo procedures were performed in accordance with EU directive 2010/63 and after approval from our institutional ethics committee (Organism for Animal Wellbeing--OPBA) and the Italian Ministry of Health (IACUC Numbers 762/2015 and 537/2017).
1. Murine Mammary Gland Collection and Digestion
<ol>
	<li>For a typical experiment, sacrifice 8-10 weeks-old virgin female mice by CO2 inhalation. Depending on the aims of the experiment, use 5-30 mice. Place the mice on dissection boards under a hood. Use needles to stretch the forelimbs and wash down the fur of the animal with ethanol.</li>
	<li>Lift the skin with forceps and perform a vertical incision starting at the level of the pelvic area and moving all the way to the cervix, leaving the peritoneum intact. To avoid rupturing the skin or the peritoneum, use round-edged scissors.</li>
	<li>Carefully detach the skin from the body with gentle movements of the scissors across the lateral axis of the body.</li>
	<li>Once the skin is fully detached from the thoracic and abdominal area, perform four incisions across the four limbs of the animal and pin down the skin with needles. Use the scissors and forceps to fully detach the body of the animal from the extended skin.</li>
	<li>Use the forceps to gently lift the mammary fat pads that are now fully exposed and carefully detach them from the skin with the aid of the scissors. Collect the lower thoracic and abdominal mammary glands of each mouse and immerse them in Dulbecco&#39;s phosphate buffered saline (DPBS), in a 50 mL conical tube. Collect up to 20 glands per tube. Keep the tissues on ice. 
	NOTE: If needed, the glands can remain on ice overnight. This is a safe stopping point. The following steps should be performed under sterile conditions.</li>
	<li>Prepare and filter the digestion medium: Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 200 U/mL collagenase and 100 &#181;g/mL hyaluronidase (see Table of Materials).</li>
	<li>Transfer up to 20 glands to a 100 mm Petri dish and mince the tissue using a scalpel or curved scissors. Avoid transferring large volumes of DPBS to the dish.</li>
	<li>Add 10 mL of digestion medium to each Petri dish and transfer the minced tissue to a 50 mL conical tube, using a 25 mL serological pipette.</li>
	<li>Seal the tubes with parafilm and place them on a rotator. Set the rotator at a low speed (0.03 x g) and incubate for 2.5 h at 37 &#176;C in a humid atmosphere containing 5% CO2.</li>
	<li>Visually inspect the suspension before proceeding to the next steps. If large pieces of tissue remain undigested, prolong the incubation at 37 &#176;C for another 30 min. 
	NOTE: As an alternative, the digestion can be performed overnight at 37 &#176;C, 5% CO2, using a gentle collagenase/hyaluronidase enzyme mix13.</li>
</ol>
2. Isolation of Primary Murine MECs
<ol>
	<li>Stop the rotator and remove the tubes from the incubator. Adjust a P1000 tip at the opening of a 5 mL serological pipette. If the suspension passes through the P1000 tip, proceed to the next steps. Otherwise, prolong the incubation at 37 &#176;C for another 30 min.</li>
	<li>Centrifuge at 100 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and resuspend the pellet of each tube in 3 mL of DPBS. 
	NOTE: The low centrifugation speed allows the removal of lymphocytes and adipose tissue cells. Gentle manipulation is required to avoid dislodging the pellet at this step.</li>
	<li>Filter the cell suspension in each tube separately, using 100, 70 and 40 &#181;m cell strainers. At each filtering step, wash the strainers with 2 mL of DPBS before collecting the pass-through. 
	NOTE: From this point on, the cell suspensions can be pooled and processed together.</li>
	<li>Centrifuge at 300 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and resuspend the cells in the remaining volume of DPBS.</li>
	<li>Proceed to the red blood cell (RBC) lysis by adding an equal volume of ammonium-chloride-potassium (ACK) lysis buffer (see Table of Materials). Mix by pipetting and incubate on ice for up to 5 min.</li>
	<li>Add 10 mL of DPBS and centrifuge at 300 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and visually inspect the pellet. If the pellet is white, proceed to the next step. Otherwise repeat the RBC lysis step (step 2.5).</li>
	<li>Resuspend the cell pellet in 1-5 mL of mammosphere media: mammary epithelial cell growth basal medium (MEBM), supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 5 &#181;g/mL insulin, 0.5 &#181;g/mL hydrocortisone, 2% B27, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL basic fibroblast growth factor (bFGF) and 0.4 IU/mL heparin (see Table of Materials).</li>
</ol>
3. Serial Mammosphere Re-plating
<ol>
	<li>For the establishment of mammosphere cultures, plate the cells onto non-tissue culture treated (ultra-low adhesion) 6-well plates at a density of 200,000 viable cells/mL in mammosphere medium. Incubate the cells for 7-10 days at 37 &#176;C, 5% CO2.</li>
	<li>Prepare and filter 1% poly-HEMA solution in 95% ethanol (see Table of Materials). Coat ultra-low adhesion 6-well plates for the following passages, by adding 400 &#181;L of 1% poly-HEMA solution per well and allowing the ethanol to dry completely. For better results, repeat the coating twice. 
	NOTE: The use of non-coated plates during the first passage allows the selective removal of fibroblasts from the culture.</li>
	<li>At the end of the 7-10 days culture, collect the primary mammospheres from all the wells and centrifuge at 300 x g, for 5 min at 4 &#176;C.</li>
	<li>Carefully decant the supernatant and proceed to the mechanical dissociation of the mammospheres using a P200 pipette with filtered tips. Pipette for approximately 100 times and visually inspect the suspension for the presence of large spheroids. 
	NOTE: A short incubation (2-5 min) of the sphere pellet in a low volume (0.2-0.5 mL) of trypsin or Accutase at 37 &#176;C, 5% CO2, can be used to facilitate mechanical dissociation. Prepare fresh mammosphere medium (step 2.7) for the plating of each passage.</li>
	<li>Resuspend in 1-5 mL of fresh mammosphere medium and count the viable cells. If applicable, split the cells in treatment groups or culture conditions.</li>
	<li>Plate 20,000 viable cells/mL on poly-HEMA-coated ultra-low adhesion 6-well plates and incubate at 37 &#176;C, 5% CO2. 
	NOTE: Mammospheres reach their maximum size within 5-7 days after cell plating. When possible, distribute the cells of each sample or condition to multiple wells to obtain technical replicates. Plating density should be kept low to avoid cell aggregation. A maximum of 20,000 cells/mL is recommended for 6-well plates and 5,000 cells/mL for 24-well plates.</li>
	<li>After 5-7 days, count the number of spheres per well. This can be done either manually, using a microscope equipped with a 10x magnification lens, or using digital image analysis (see step 4).</li>
	<li>Collect the mammospheres of each well separately and centrifuge at 300 x g, for 5 min at 4 &#176;C.</li>
	<li>Carefully decant the supernatant and proceed to the mechanical dissociation of the mammospheres using a P200 pipette with filtered tips. Pipette for approximately 100 times and visually inspect the suspension for the presence of large spheroids.</li>
	<li>Resuspend in 1 mL of fresh mammosphere medium and count the viable cells. Pool the cells of each sample or condition and repeat steps 3.6-3.10. Record the number of cells plated and the number of spheres and cells counted per well for each passage. Proceed to step 5 for the cumulative growth calculation.</li>
</ol>
4. Sphere Enumeration Using Digital Image Analysis (DIA)
<ol>
	<li>At the end of each passage, scan the entire surface of all the wells and acquire images of the spheres using a digital camera mounted on a stereoscope. Save the images as .tif files.</li>
	<li>Import the stereoscope images as an Image Sequence using ImageJ14. Set the scale and the type of image to 8-bit.</li>
	<li>Duplicate the stack of images and select Subtract Background from the tab Process on the menu bar. Check the options Light background and Sliding paraboloid and click on the button OK. Process all the images of the stack.</li>
	<li>Select Adjust and then Threshold from the tab Image of the menu bar. By clicking on Apply, a dialogue window titled Convert Stack to Binary will appear. Select Default as method and Light as background. Check the box Calculate threshold for each image and click on the button OK.</li>
	<li>Select sequentially the commands Watershed, Open and Erode from the Binary list under the tab Process of the menu bar. Process all images of the stack by clicking on the button Yes of the dialogue box that appears. 
	NOTE: These processes allow for visual segmentation of objects that touch, object smoothing and removal of pixels from the edge of the objects.</li>
	<li>Select the function Analyze Particles from the menu Analyze. Set the minimum size threshold at 10,000 &#181;m2 and circularity between 0.50 and 1.00. Select the option Ellipses from the Show drop-down menu. Check Summarize, Exclude on edges and In situ Show and click on the button OK. Process all images of the stack.</li>
	<li>Visually inspect the correspondence of the ellipses with spheres and, if needed, correct the particle count accordingly. Sum the counts from all the frames of each well to obtain the total count of mammospheres per well.</li>
</ol>
5. Cumulative Growth Curve Calculation
NOTE: The number of mammospheres counted in each well at the end of each passage (PN) reflects the number of mammosphere-initiating cells seeded at the beginning of PN.
<ol>
	<li>For each passage (PN), register the number of plated cells and the number of cells and spheres counted per well. Calculate the sphere size at the end of each passage: 
	sphere size PN (cells / sphere) = cells counted PN / spheres counted PN 
	NOTE: The sphere size at PN is a measure of the proliferative potential of each mammosphere-initiating cell seeded at PN.</li>
	<li>Infer the number of plated spheres by dividing the number of cells plated for PN by the sphere size calculated at the end of the previous passage (PN-1): 
	spheres plated PN = cells plated PN / sphere size PN-1 
	NOTE: By convention, the sphere size is assumed stable during the first passage of the culture (i.e., sphere size P0 = sphere size P1). If multiple wells are used as technical replicates, use the average sphere size at PN-1 as denominator.</li>
	<li>Calculate the cumulative cell and sphere number for each well per passage: 
	cumulative number PN = (count PN / plated PN) X cumulative number PN-1. 
	NOTE: By convention, cumulative number P0 = plated P1. If multiple wells are used as technical replicates, calculate the average cumulative number per sample or condition for each passage.</li>
	<li>Plot the data points on a semi-logarithmic scale. Display the passage number (P0 to PN) on the x axis (linear scale) and the cumulative cell or sphere number on the y axis (logarithmic scale).</li>
	<li>Fit an exponential trend-line to the data points and calculate the coefficient of determination (R2) to measure the goodness of the fit. 
	NOTE: The trend-line fitted to the data points should approximate an exponential curve, as expected for a cell population that grows or dies with a constant rate. R2 takes values between 0 and 1, with values closest to 1 indicating a better fit.</li>
	<li>Depict the equation of the trend-line as a natural exponential function to infer the growth rate (GR) of the culture: 
	y = y0 e(GR)x, where y0 is the value of y when x = 0.</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a protocol for the quantitative description of MaSC growth properties in vitro. As an example, we present the effect of low-level constitutive Myc expression on normal murine MaSCs. This approach, however, can be equally applied to various contexts. Human or murine primary cells, as well as established cell lines, can be cultured in anchorage independent conditions to establish mammosphere cultures that can be serially passaged. Gene overexpression and RNA interference can be easily introduced in the protocol with the addition of a viral transduction step at the end of the first passage (after step 3.5). Alternatively, cells can be infected in adhesion and then plated as mammospheres.
A critical aspect of the assay presented here is the seeding cell density, which should be low enough to avoid the generation of aggregates interfering with the interpretation of the results16,17. The morphology of the mammospheres can be informative to resolve this ambiguity. Only compact, round spheres should be enumerated at the end of each passage. Both the circularity of the spheroids and the size should be taken into consideration. Using the automated process of the DIA, this step is ensured with the appropriate thresholds in an objective and absolute manner. Often, progenitors will form acinar structures or smaller clusters of cells which should be excluded from the mammosphere counts. As a rule of thumb, we use a threshold of 100 &#181;m diameter. Finally, care should be taken to avoid the transfer of intact or non-fully dissociated mammopsheres from one passage to the next. On the other hand, excess pipetting will lead to increased cell death. Thus, if such difficulties are encountered, we recommend using mild trypsinization or Accutase treatment and passing the dissociated spheres through a 40 &#181;m strainer to ensure the generation of single-cell suspensions.
Sphere forming efficiency (SFE) has been used alternatively, as a surrogate for SC or CSC quantitation ex vivo in mammosphere cultures. SFE is indeed a measure of stem-like cells in a given cell population. However, it represents a less conscientious approach since it provides information only at distinct time points. The calculation of cumulative sphere numbers and the generation of cumulative growth curves, instead, enables the inference of the growth rate of the culture from the initial cell seeding step until the culture exhausts or, in the case of immortalized cultures, for the desired number of passages. The assessment of growth properties allows the evaluation of the deviation from the exponential growth through the coefficient R2 and, at a second step, the assessment of the GR value itself.
Importantly, cumulative mammosphere growth curves can be used to evaluate the effect of small molecule inhibitors or other chemotherapeutic drugs selectively at the CSC level6,11. Contrary to normal primary mammospheres, which functionally exhaust in 5-7 passages, tumor mammospheres tend to expand indefinitely. This feature is linked to the unlimited CSC self-renewal ability. The effects on proliferation and CSC self-renewal can be uncoupled through the generation of tumor cell and mammosphere growth curves, respectively. A CSC-specific effect is expected to result in a decrease in the cumulative mammosphere growth rate, with or without effect on the cumulative cell growth rate6,11.
Finally, another area of interest is the one of adult tissue SC reprogramming. Fully grown mammospheres consist of a phenotypically heterogeneous cell population, in which only a minor fraction retains stem-like features, including mammosphere-initiating ability and mammary gland regeneration upon transplantation in vivo6,9,11,18,19. Mammary progenitors can be thus isolated either using in vitro label-retaining assays6,9,11 or, ex vivo, using established surface markers2,3. Notably, mammary progenitors do not survive anoikis and are unable to form mammospheres. Enforced Myc expression has been shown to confer mammosphere initiation potential to mammary progenitors isolated as PKHneg11, resulting in the generation of a culture that can be passaged indefinitely. Similarly, interference of negative regulators of physiological reprogramming can be tested using the same assay. In this context, a common issue that may arise is the limited number of cell input. If the cell input is lower than 10,000 cells, we recommend seeding in 24-well plates (maximum 5,000 viable cells/mL). Nevertheless, anchorage independent culture conditions can be proven to be too harsh for scoring reprogramming, especially in cases where the reprogramming effect is not immediate. In such cases, the use of a supportive matrix and 3-dimensional organoid cultures could be more appropriate20.
Overall, the mammosphere assay is a cost-effective option that can be easily employed for scoring stem-like properties in normal and tumoral MEC populations. The quantitative approach taken in this protocol facilitates the comparisons between cultures carried out in different conditions or exposed to diverse stimuli. When followed rigorously, it provides a relatively simple ex vivo model system that permits uncoupling of the multiple players that define stem properties in vivo, offering the possibility of more detailed mechanistic studies.

Isolation and in vitro culture of mammary epithelial stem and progenitor cells have become essential for understanding their properties in mammary cell biology. Elegant lineage tracing and serial transplantation assays have enabled the study of stem cells (SCs) and other tissue subsets in the context of their in vivo niche. However, this approach is time consuming and requires the generation of reporter mouse models1,2,3,4,5. Therefore, in vitro culture and propagation of mammary stem cells (MaSCs) while sparing key stemness features, namely self-renewal and differentiation ability, is one of the biggest challenges in the field. In the last years, the mammosphere assay has been widely used to model both normal mammary tissue and breast cancer growth, to quantify normal or cancer SCs (CSCs) and assess their self-renewal ability as a surrogate reporter of their activity in their respective in vivo context6,7,8,9,10,11.
The mammosphere assay is an efficient and cost-effective approach, in which freshly isolated mammary epithelial cells (MECs) are cultured in non-adherent conditions, with the premise that only MaSCs will survive and form spheres in suspension while all the other cell types will die by anoikis. Moreover, the ability to form several generations of mammospheres in serial non-adherent passages is related to the self-renewal ability of the MaSCs6,9,11. Here, we describe a detailed protocol of a quantitative mammosphere assay, which was initially developed by Dontu and colleagues7 as a modification of the pioneering neurosphere assay12, enabling the growth of putative SCs in non-adherent, serum-free conditions with the addition of appropriate growth factors7,12.

<table><tbody><tr><td>ACK lysis buffer</td><td>Lonza</td><td>10-548E</td><td>Ammonium-chloride-potassium lysis bufer, 100 mL.</td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504-044</td><td>B27 supplement 50X (10 mL). Final concentration 2% v/v.</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>Human recombinant fibroblast growth factor - basic, 50 &amp;mu;g. Stock solution 100 &amp;mu;g/&amp;mu;L in Tris 5 mM pH 7.6. Final dilution 0.02% v/v.</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C2674</td><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt;. Type I-A, lyophilized powder, 1 g. Stock 20,000 U/mL in DMEM. Final dilution 1% v/v.</td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-614F</td><td>Dulbecco&#039;s modified Eagle&#039;s medium</td></tr><tr><td>DPBS</td><td>Microgem</td><td>S17859L0615</td><td>Dulbecco&#039;s phosphate buffered saline</td></tr><tr><td>EGF</td><td>Tebu-Bio</td><td>AF-100-15</td><td>Recombinant human epithelial growth factor. Stock solution 100 &amp;mu;g/mL in sterile dH&lt;sub&gt;2&lt;/sub&gt;O. Final dilution 0.02% v/v.</td></tr><tr><td>Glutamine</td><td>Lonza</td><td>17-605E</td><td>L-Glutamine, 200 mM. Final dilution 1% v/v.</td></tr><tr><td>Heparin</td><td>PharmaTex</td><td>34692032</td><td>Stock concentration 5,000 IU/mL. Final dilution 0.008% v/v.</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H4272</td><td>Type IV-S, powder, 750-3,000 U/mg solid, 30 mg. Stock solution 10 mg/mL in sterile dH&lt;sub&gt;2&lt;/sub&gt;O. Final dilution 1% v/v.</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888</td><td>Stock concentration 100 &amp;mu;g/mL. Final dilution 0.5% v/v.</td></tr><tr><td>Insulin</td><td>SAFCBiosciences</td><td>91077C</td><td>Insulin, human recombinant, dry powder, 250 mg. Stock concentration 1 mg/mL. Final dilution 0.5% v/v.</td></tr><tr><td>Low attachment 6-well plates</td><td>Corning</td><td>351146</td><td>Sterile 6-well not treated cell culture plates with clear flat bottom and lid.</td></tr><tr><td>MEBM</td><td>Lonza</td><td>CC-3151</td><td>Mammary epithelial cell growth basal medium</td></tr><tr><td>Penicllin-Streptomycin mixture</td><td>Lonza</td><td>17-602F</td><td>Contains 10,000 U potassium penicillin and 10,000 &amp;micro;g streptomycin sulfate per mL in 0.85% saline. Final dilution 1% v/v.</td></tr><tr><td>Poly-HEMA</td><td>Sigma</td><td>P3932</td><td>Dissolve in 95% EtOH overnight at 55 &amp;deg;C. Stock concentration 12% w/v. Final dilution 1% v/v in 95% EtOH. Filter (0.22 &amp;mu;m) before use.</td></tr></tbody></table>

quantification of self-renewal in murine mammosphere cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Myc overexpression in normal MECs, leads to an increased frequency of mammosphere initiating cells. This is achieved through a double mechanism: Myc increases the rate of MaSC symmetric divisions and the frequency of progenitor reprogramming into new MaSCs11. To test the effect of low constitutive Myc expression, we used the Rosa26-MycER transgenic mouse model, in which Myc activity can be induced by 4-hydroxytamoxifen (4-OHT)15. We first plated the MECs on ultra-low adhesion 6-well plates to remove fibroblasts, in the absence of 4-OHT. After the first passage, we split the culture in two: two wells were kept untreated (control) and two wells were treated with 200 nM 4-OHT (MycER). We counted the sphere and cell numbers of 5 consecutive passages for three independent experiments (Table 1). The cumulative cell and sphere numbers per passage are shown on Table 2. Induction with 4-OHT leads to increased sphere and cell growth rates, as shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60256/60256fig1a.jpg" /> 
Figure 1: Representative results. Cumulative sphere (A) and cell (B) growth curves of control and MycER mammospheres. Mean and standard deviation of 3 independent experiments are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60256/60256fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="3">1st plating</td>
			<td colspan="3">2nd plating</td>
			<td colspan="3">3rd plating</td>
			<td colspan="3">4th plating</td>
			<td colspan="3">5th plating</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
		</tr>
		<tr>
			<td rowspan="4">Exp1</td>
			<td rowspan="2">Control</td>
			<td>77,000</td>
			<td>50,000</td>
			<td>31</td>
			<td>65,000</td>
			<td>58,500</td>
			<td>41</td>
			<td>57,000</td>
			<td>30,000</td>
			<td>32</td>
			<td>30,000</td>
			<td>22,000</td>
			<td>5</td>
			<td>22,000</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>77,000</td>
			<td>81,000</td>
			<td>41</td>
			<td>65,000</td>
			<td>55,000</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>77,000</td>
			<td>31,0000</td>
			<td>193</td>
			<td>80,000</td>
			<td>375,000</td>
			<td>323</td>
			<td>80,000</td>
			<td>380,000</td>
			<td>217</td>
			<td>80,000</td>
			<td>220,000</td>
			<td>223</td>
			<td>80,000</td>
			<td>170,000</td>
			<td>155</td>
		</tr>
		<tr>
			<td>77,000</td>
			<td>110,000</td>
			<td>142</td>
			<td>80,000</td>
			<td>505,000</td>
			<td>396</td>
			<td>80,000</td>
			<td>270,000</td>
			<td>149</td>
			<td>80,000</td>
			<td>290,000</td>
			<td>194</td>
			<td>80,000</td>
			<td>250,000</td>
			<td>160</td>
		</tr>
		<tr>
			<td rowspan="4">Exp2</td>
			<td rowspan="2">Control</td>
			<td>75,000</td>
			<td>75,000</td>
			<td>71</td>
			<td>60,000</td>
			<td>17,000</td>
			<td>34</td>
			<td>28,000</td>
			<td>45,000</td>
			<td>29</td>
			<td>45,000</td>
			<td>13,000</td>
			<td>2</td>
			<td>13,000</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>75,000</td>
			<td>47,000</td>
			<td>45</td>
			<td>60,000</td>
			<td>11,000</td>
			<td>47</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>75,000</td>
			<td>200,000</td>
			<td>188</td>
			<td>80,000</td>
			<td>225,000</td>
			<td>277</td>
			<td>80,000</td>
			<td>230,000</td>
			<td>155</td>
			<td>80,000</td>
			<td>210,000</td>
			<td>211</td>
			<td>80,000</td>
			<td>100,000</td>
			<td>95</td>
		</tr>
		<tr>
			<td>75,000</td>
			<td>250,000</td>
			<td>192</td>
			<td>80,000</td>
			<td>202,500</td>
			<td>283</td>
			<td>80,000</td>
			<td>305,000</td>
			<td>185</td>
			<td>80,000</td>
			<td>160,000</td>
			<td>237</td>
			<td>80,000</td>
			<td>100,000</td>
			<td>133</td>
		</tr>
		<tr>
			<td rowspan="4">Exp3</td>
			<td rowspan="2">Control</td>
			<td>82,500</td>
			<td>130,000</td>
			<td>121</td>
			<td>80,000</td>
			<td>45,000</td>
			<td>105</td>
			<td>80,000</td>
			<td>110,000</td>
			<td>86</td>
			<td>80,000</td>
			<td>58,500</td>
			<td>75</td>
			<td>58,500</td>
			<td>58,500</td>
			<td>78</td>
		</tr>
		<tr>
			<td>82,500</td>
			<td>125,000</td>
			<td>177</td>
			<td>80,000</td>
			<td>71,250</td>
			<td>42</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>82,500</td>
			<td>325,000</td>
			<td>457</td>
			<td>80,000</td>
			<td>610,000</td>
			<td>327</td>
			<td>80,000</td>
			<td>367,000</td>
			<td>309</td>
			<td>80,000</td>
			<td>500,000</td>
			<td>260</td>
			<td>80,000</td>
			<td>115,000</td>
			<td>146</td>
		</tr>
		<tr>
			<td>82,500</td>
			<td>475,000</td>
			<td>463</td>
			<td>80,000</td>
			<td>455,000</td>
			<td>392</td>
			<td>80,000</td>
			<td>415,000</td>
			<td>204</td>
			<td>80,000</td>
			<td>470,000</td>
			<td>295</td>
			<td>80,000</td>
			<td>185,000</td>
			<td>161</td>
		</tr>
	</tbody>
</table>
Table 1: Numbers of spheres counted and numbers of cells plated and counted at each passage.
<img alt="Table 2" class="xfigimg" src="/files/ftp_upload/60256/60256table2.jpg" /> 
Table 2: Calculation of plated sphere numbers and cumulative sphere and cell numbers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60256/Table2.xlsx" target="_blank">Please click here to view a larger version of this table. (Right-click to download.)</a>

Watch this Scientific Journal Video about Quantification of Self-renewal in Murine Mammosphere Cultures at JoVE.com

Quantification of Self-renewal in Murine Mammosphere Cultures

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

quantification-of-self-renewal-in-murine-mammosphere-cultures

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.3K Views.  European Institute of Oncology IRCCS. Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

Video: Quantification of Self-renewal in Murine Mammosphere Cultures

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

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.

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 ...

Cancer Research

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 ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

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

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Medicine

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

Quantification of Self-renewal in Murine Mammosphere Cultures

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In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

De Novo Generation of Somatic Stem Cells by YAP/TAZ