Name: co-culture and transduction of murine thymocytes on delta-like 4-expressing stromal cells to study oncogenes in t-cell leukemia
Uploaded: 2023-06-09T15:00:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia at JoVE.com

This work was supported by the intramural program of the National Cancer Institute, project ZIABC009287. OP9-DL4 was obtained from Dr. Juan Carlos Z&#250;&#241;iga-Pfl&#252;cker (Sunnybrook Health Sciences Centre, Toronto, ON, Canada). The authors thank the NCI-Frederick Laboratory Animal Sciences Program for their continued technical assistance and experimental advice and input, as well as Jeff Carrel, Megan Karwan, and Kimberly Klarmann for flow cytometry assistance. We are grateful to Howard Young for critical advice and input.

All animal experiments described were approved by the NIH Institutional Biosafety Committee (IBC) and Animal Care and Use Committee (ACUC). See the Table of Materials for details related to all the reagents and materials used in this protocol. Refer to published guidelines19&#160;for more details on retrovirus-producer cell culture and maintenance procedures. See Figure 1 for an overview of the protocol.
1. Starting the culture of OP9-DL4 cells (Day 1)
<ol>
	<li>Thaw OP9-DL4 cells rapidly by holding the vial and gently shaking in a water bath at 37 &#176;C. Immediately transfer the cells to a centrifuge tube containing 5 mL of the OP9 medium (MEM-alpha medium, 10% FBS, 50 U/mL penicillin/streptomycin, 55 &#181;M 2-mercaptoethanol, 2 mM L-glutamine) to remove the cryoprotectant agents.</li>
	<li>Centrifuge at 300 &#215; g for 5 min at room temperature. Discard the supernatant, and resuspend the cells in 1 mL of OP9 medium.</li>
	<li>Place 5 mL of OP9 medium into a T25 flask, and add the resuspended cells to this flask. Culture at 37 &#176;C and 5% CO2.</li>
	<li>After 2&#8211;3 days, subculture the cells using a 1:3 split (passage the cells every Monday, Wednesday, and Friday). 
	NOTE: Split the OP9-DL4 cells before they reach confluency. The OP9-DL4 flask must be 80%&#8211;90% confluent on days 6&#8211;7 and on days 8&#8211;9+ for co-culture with thymocytes. Hence, it is important to plan how many OP9-DL4 cells will be required on those days while splitting the OP9-DL4 cells.
	<ol>
		<li>Discard the medium from the flask, and wash the monolayer with 1x PBS. Discard the PBS, add 1 mL of 0.25% trypsin, and incubate for 1&#8211;5 min at 37 &#176;C, or until the cells have dislodged from the flask. Thump the flask to dislodge the cells. 
		NOTE: (Optional) The progress of cell dissociation can be checked by microscopy.</li>
		<li>Add 5 mL of OP9 medium to inactivate the trypsin, and resuspend the cells by rinsing the cell-covered surface of the flask during resuspension.</li>
		<li>Centrifuge the cell suspension at 300 &#215; g for 5 min at room temperature and resuspend in 3 mL of OP9 medium (for a 1:3 split).</li>
		<li>Place 1 mL of the cell suspension into a T25 flask that already contains 5 mL of fresh OP9 medium. Freeze/discard the remaining cells.
		<ol>
			<li>To freeze the OP9-DL4 cells, resuspend the cells in 90% FBS/10% DMSO at a ratio of 1 mL of freezing medium to one vial of cells.</li>
			<li>Freeze in 1 mL cryovials at &#8722;80 &#176;C or in liquid nitrogen, depending on the future requirements. Store in liquid nitrogen for prolonged storage.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Starting the culture of the retrovirus-producer cell line (Day 1)
<ol>
	<li>Thaw the retrovirus-producer cell line rapidly by holding the vial and gently shaking in a water bath at 37 &#176;C. Immediately transfer the cells to a centrifuge tube containing 5 mL of retrovirus-producer cell medium (RPC: DMEM, 10% FBS) to remove the cryoprotectant agent.</li>
	<li>Centrifuge at 300 &#215; g for 5 min at room temperature. Discard the supernatant, and resuspend the cells in 1 mL of RPC medium.</li>
	<li>Place 5 mL of RPC medium into a T25 flask, and add the resuspended cells to this flask. Culture at 37 &#176;C and 5% CO2.</li>
	<li>Passage the cells every 2&#8211;3 days at a 1:5 to 1:8 split.
	<ol>
		<li>Thump the flask to dislodge the cells, and resuspend the cells by pipetting aggressively. 
		NOTE: These cells can be removed from the flask surface by aggressive pipetting without trypsin. Alternatively, trypsinize (0.05% trypsin/0.53 mM EDTA) until the cells easily detach and can be readily pipetted into a single-cell suspension.</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 300 &#215; g for 5 min, and resuspend in 5 mL of RPC medium (for a 1:5 split).</li>
	<li>Place 1 mL of the cell suspension into a T25 flask that already contains 5 mL of fresh RPC medium. Freeze/discard the remaining cells. Freeze the retrovirus-producer cells in the same way as described for OP9-DL4 cells (steps 1.4.4.1&#8211;1.4.4.2).</li>
</ol>
3. Beginning the culture of the retrovirus-producer cells in 6-well plates (Days 4&#8211;5)
<ol>
	<li>Seed the retrovirus-producer cells 18&#8211;24 h prior to transfection at 70%&#8211;90% confluency in each well of a 6-well tissue culture plate in 2 mL of RPC medium,and incubate at 37 &#176;C and 5% CO2. 
	NOTE: Transfect two wells of the retrovirus-producer cells for every 2&#8211;5 &#215; 105 to 1 &#215; 106 thymocytes that will be transduced.</li>
</ol>
4. Transfecting the retrovirus-producer cells to generate the retrovirus containing the genes of interest (Days 5&#8211;6)
<ol>
	<li>Replace the medium with fresh RPC medium 1 h before transfection.</li>
	<li>Prepare lipofection mixtures (for each well of a 6-well plate&#8212;scale up as needed): Dilute 4 &#181;g of DNA (2 &#181;g of helper plasmid (pCL-Eco) and 2 &#181;g of transfer plasmid (pMIG) in 250 &#181;L of reduced-serum medium. Mix gently.</li>
	<li>Mix 10 &#181;L of transfection reagent with 250 uL of the reduced-serum medium from step 4.2. Incubate for 5 min at room temperature.</li>
	<li>After 5 min of incubation, combine the diluted DNA with diluted transfection reagent (total volume = 500 &#181;L). Mix gently, and incubate for 20&#8211;25 min at room temperature.</li>
	<li>Add the 500 &#181;L of DNA/transfection reagent mixtures gently to the well containing the retrovirus-producer cells by dropping onto the cells with a circular movement. Mix gently by rocking the plate back and forth, and incubate the plate in a 37 &#176;C incubator for 16&#8211;24 h. 
	NOTE: Subconfluent cells (80%&#8211;90%) are best suited for transfection and potentially generate the highest viral titer. As the retrovirus-producer cells dislodge very easily from the flask, avoid sudden movements when handling these cells.</li>
</ol>
5. Changing the retrovirus-producer cell medium (Days 6&#8211;7)
<ol>
	<li>Replace the old medium approximately 16&#160;h after transfection with 2 mL of fresh RPC medium. Continue to incubate cells at 37 &#176;C for 20&#8211;24 h.</li>
	<li>Evaluate the transfection efficiency under a fluorescence microscope (optional). 
	NOTE: In this protocol, GFP-positive cells are the cells of interest (see Figure 2). This step depends on the retrovirus vector selected to use in this protocol (whether a reporter gene is present in the backbone).</li>
</ol>
6. Thymocyte preparation and depletion of CD4+&#160;and&#160;CD8+&#160;cells (Days 6&#8211;7)
<ol>
	<li>Harvest the thymus from a 4&#8211;6 week old C57BL/6 mouse. For more details on thymus harvesting, refer to Xing and Hogquist20. 
	NOTE: Mice were euthanized by CO2 inhalation followed by cervical dislocation.</li>
	<li>Prepare a thymocyte single-cell suspension by placing the thymus in 5 mL of PBS in a Petri dish. Using sterile glass slides, place the thymus between the frosted surfaces of the slides, and gently rub the slides together, rolling the thymus between the two slides. Rinse the glass slides to collect the cells, and discard the remnant thymic stromal tissue. 
	NOTE: The estimated yield from one thymus is 90 &#215; 106&#8211;100 &#215; 106 cells, and approximately 1% of the cells will remain after CD4 and CD8 depletion. Use thymus tissue from younger mice for a better yield of cells, since the cellularity of the mouse thymus in the first weeks after birth increases quickly, reaches a plateau at 4&#8211;6 weeks of age, and progressively involutes subsequently21. Count the cells using an automatic blood cell counter or any alternative method.</li>
	<li>Filter the thymic suspension through a 30 &#181;m or 40 &#181;m filter by pipetting 5 mL of the thymic suspension through a cell strainer. Centrifuge the cells at 300 &#215; g for 10 min.</li>
	<li>Remove the supernatant, and lyse the red blood cells with ACK lysis buffer by adding (depending on the sample size) 1 mL of buffer per tube for 1 min. Add 5 mL of cell depletion buffer (500 mL of PBS [pH 7.2], 0.5% BSA, 2 mM 0.5M EDTA, pH 8.0) to deactivate the ACK lysis buffer, centrifuge at 300 &#215; g for 10 min, and resuspend in 1&#8211;5 mL of cell depletion buffer for counting. 
	NOTE: Set aside the following on ice before depletion (pre-depletion): 70 &#181;L of cell suspension to count (vary this depending on the counting cell method chosen) and 200 &#181;L of cell suspension to determine the efficiency of depletion by staining for CD4 and CD8 and analyzing by flow cytometry22,23.</li>
	<li>Count the cells, and centrifuge at 300 &#215; g for 10 min. Resuspend the cells at 1 &#215; 107/80 &#181;L of cell depletion buffer. Add 10 &#181;L each of CD4 and CD8 microbeads per 1 &#215; 107 cells. Mix well, and incubate for 15 min in the dark in the refrigerator (2&#8211;8 &#176;C).</li>
	<li>Prepare a depletion column by rinsing it with 2 mL of depletion buffer and discarding the flowthrough.</li>
	<li>Wash the cells from step 6.6 by adding 1&#8211;2 mL of depletion buffer per 1 &#215; 107 cells, and centrifuge at 300 &#215; g for 10 min. Remove and discard the supernatant.</li>
	<li>Resuspend up to 1.25 &#215; 108 cells in 500 &#181;L of depletion buffer. Apply the cell suspension to the column, and collect the flowthrough (unlabeled cells). Wash the column 2x with 1 mL of buffer, and collect the flowthrough. 
	NOTE: Only add new buffer when the column reservoir is empty. Set aside the following on ice after depletion (post-depletion control): 70 &#181;L of cell suspension to count (vary this depending on the counting cell method chosen) and 1,000 &#181;L of cell suspension to determine the efficiency of depletion by staining for CD4 and CD8 and analyzing by flow cytometry22,23.</li>
	<li>Stain the depletion efficiency controls by splitting the 200 &#181;L of cells collected before depletion into four FACS tubes: unstained, CD4 single-stained, CD8 single-stained, and CD4/CD8 double-stained (use the unstained and single-stained samples to set up the flow cytometry parameters). Use the 1,000 &#181;L of cells collected after depletion to stain for CD4 and CD8 and compare with the double-stained sample collected before depletion (see typical depletion results in Figure 3). 
	NOTE: Adjust the volumes for flow cytometry staining according to the antibody manufacturer&#8217;s recommendation (e.g., 1 &#181;L of anti-CD4-FITC + 0.5 &#181;L of anti-CD8-PE per 1 x 106 cells in 50 &#181;L of buffer).</li>
</ol>
7. Culturing thymocytes on OP9-DL4 cells (Days 6&#8211;7)
<ol>
	<li>Place 2&#8211;5 &#215; 105 to 1 &#215; 106 post-depletion thymocytes into a T25 flask of 80%&#8211;90% confluent OP9-DL4 cells in OP9 medium containing cytokines (10 ng/mL recombinant IL-7 and recombinant hFLT-3). Culture at 37 &#176;C and 5% CO2. Grow for approximately 24 h on OP9-DL4 cells. 
	NOTE: This duration is required to make the T cells transducible (see a typical result in Figure 4). Reserve one T25 flask of the co-culture of thymocytes and OP9-DL4 cells to be used as a control (untransduced). The untransduced thymocytes will be stained together with the transduced thymocytes (step 9.1) as a negative control to evaluate the transduction efficiency. Untransduced thymocytes can also be used to measure the effect of the transgene expression on cell differentiation. Discard the cytokine-containing medium after 1 month.</li>
</ol>
8. Harvesting the retrovirus from the supernatant and using it to transduce the thymocytes (Days 8&#8211;9)
<ol>
	<li>Collect the supernatant containing the retroviruses from the transfected cells by tilting the 6-well plate and positioning a 3&#8211;5 mL syringe at the bottom of the plate while pulling the plunger to aspirate the supernatant. Replace the medium with 2 mL of fresh RPC medium. Continue to incubate the cells at 37 &#176;C for 20&#8211;24 h for the second transduction in step 8.9.</li>
	<li>Filter the retrovirus supernatant through a 0.45 &#181;m syringe filter, and collect the filtrate in a 50 mL tube. 
	NOTE: Do not freeze the retroviral supernatant. Use a freshly made virus preparation for transduction.</li>
	<li>Collect thymocytes from the OP9-DL4 culture by aggressive pipetting to remove the thymocytes and OP9-DL4 cells from the flask surface. Filter the cell suspension through a 40 &#181;m cell strainer to remove most of the OP9-DL4 cells, and collect the filtrate in a 50 mL tube. 
	NOTE: OP9 cells are very adherent. Although aggressive pipetting may remove some of the OP9-DL4 cells from the flask, the disruption of the OP9-DL4 monolayer during this process is minimal, and the OP9-DL4 cells that come off will be filtered out with the 40 &#181;m cell strainer, since the OP9-DL4 cells are much larger than the primary thymocytes. If the OP9-DL4 cells are still 80%&#8211;90% confluent, remove the thymocytes, and re-plate them in the same OP9-DL4 flask. Alternatively, a new OP9-DL4 flask must be prepared.</li>
	<li>Centrifuge the thymocytes in the filtrate from step 8.3 at 300 &#215; g for 5 min. Discard the supernatant.</li>
	<li>In the 50 mL tube, resuspend the thymocytes in 0.5&#8211;1 mL of OP9 medium + cytokines (see step 7.1). Add 1&#8211;2 mL of RPC medium containing the virus (twice as much RPC medium as thymocyte medium). Add hexamethrine bromide (10 &#181;g/&#181;L stock concentration) to yield 8 &#181;g of hexamethrine bromide/mL of total cell suspension.</li>
	<li>Spinoculate by centrifuging the cells at 850 &#215; g for 1 h at room temperature.</li>
	<li>Resuspend the cells in 6 mL of OP9 medium + cytokines per flask (see step 7.1), and add the suspension back onto the OP9-DL4 cell monolayer. 
	NOTE: Alternatively, have new OP9-DL4 flasks ready to receive the transduced thymocytes. If the thymocytes are to be placed back into a used OP9-DL4 flask, make sure to add fresh OP9 medium to the OP9-DL4 cells during the 1 h of thymocyte spinoculation to keep the cells healthy and ensure 80%&#8211;90% confluency.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
	<li>Repeat steps 8.2&#8211;8.7 using a new well of the retrovirus-containing cell supernatant.</li>
</ol>
9. Maintaining transduced thymocytes on OP9-DL4 culture for 2&#8211;5 days or freezing as needed (Day 9+)
<ol>
	<li>Assess the thymocyte differentiation by flow cytometry by staining thymocytes for T cell development markers such as CD4, CD8, CD25, and CD4423. See typical thymocyte differentiation results in Figure 5.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ThPOK+in+control+of+CD4/CD8+lineage+commitment.">The role of ThPOK in control of CD4/CD8 lineage commitment.</a> Annual Review of Immunology. 28, 295-320 (2010).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+CXXC+motif+in+CD3epsilon+is+critical+for+T+cell+development+and+TCR+signaling.">A conserved CXXC motif in CD3epsilon is critical for T cell development and TCR signaling.</a> PLoS Biology. 7 (12), e1000253 (2009).</li><li>Aliahmad, P., Kadavallore, A., de la Torre, B., Kappes, D., Kaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOX+is+required+for+development+of+the+CD4+T+cell+lineage+gene+program.">TOX is required for development of the CD4 T cell lineage gene program.</a> Journal of Immunology. 187 (11), 5931-5940 (2011).</li></ol>

The authors have no conflicts of interest to disclose

The protocol described here was developed specifically for thymus-derived DN (CD4&#8722;/CD8&#8722;) T cell studies with retroviral transfection followed by an OP9-DL4 differentiation model. However, it is likely that the target cells that will be subjected to this protocol of transduction followed by cell differentiation will have a wider interdisciplinary utility. Thus, in addition to immature thymocytes, hematopoietic stem cells, such as cells derived from either fetal liver or bone marrow, could potentially be used in this protocol.
The OP9-DL4 system has proven to be an effective model to study gene function in a variety of aspects, including cell differentiation17 and oncogenesis15. While the retroviral modification of hematopoietic progenitors is a well-established technique that enables stable genetic modification, combining the induction of cell differentiation on the OP9-DL4 system and retroviral transduction requires care and skill. The critical aspect for achieving success with this protocol is ensuring all the steps are well-coordinated, since the protocol involves using three different cell types that need to be kept healthy and at the ideal confluence required for each specific stage. With that in mind, it is important to perform all the quality control checkpoint analyses after the execution of each step before proceeding to the next step. This will ensure that all the steps are working. Therefore, checking the depletion, transfection, and transduction efficiencies is important for the successful execution of this protocol (see a typical result for transduction efficiency in Figure 4). Good primary cell transduction efficiency is linked to a high viral titer. Typically, larger inserts result in lower virus titers18. For training purposes, we use an empty vector to represent the results that can be obtained with this protocol. In our experience, the transduction and transfection efficiencies vary according to the insert size, especially considering the viral backbone-expressing reporter genes, such as GFP. One strategy that can be used when studying the interaction of more than one gene is to clone each gene into a different transfer vector, followed by individual virus production, and finally, co-transduction of the target cell. In that case, a selection step may be applied to eliminate the singly transduced cells and retain only the co-transduced cells.
It is worth noting that the majority of OP9-DL1/DL4 stromal feeder layer cell lines have been genetically engineered to express GFP as part of the DL1 or DL4 constructs7. In this protocol, we used a retroviral vector that also expresses GFP; however, it is brighter than the GFP protein expressed by OP9-DL4 cells and does not interfere with the transduction inspection when visualizing the co-culture under the fluorescence microscope.
OP9 cells differentiate into adipocytes after many passages, long periods in culture, or under conditions of over-confluency19. This is evidenced by the development of large vacuoles. Thus, OP9 cells presenting these characteristics should not be used in this protocol. Transfecting overly confluent retrovirus-producer cells will result in a low virus titer. Indeed, the subconfluent stage is when the cells are most transfectable. Furthermore, transfecting low-confluence retrovirus-producer cells will decrease the cell stress in the transfection process and give the highest virus titer.
While, in this protocol, we do not titrate the virus supernatant, virus supernatant titration must be considered in some cases, such as in the absence of a reporter gene in the retroviral vector, which would prevent the indirect determination of viral production, or in cases where the experimental design requires a more accurate number of the transgene copies to be integrated into the target cell genome. However, it is important to note that the titer of retroviral vector supernatant decreases significantly when stored at &#8722;80 &#176;C or 4 &#176;C until the titration results are obtained. Therefore, using freshly prepared virus supernatant for transduction will yield better transduction efficiency.
The thymus contains a large number of double-positive (DP) thymocytes (more than 85%) and about 10% single-positive15&#160;cells&#160;(CD4 or CD8), which are the post-DN stage thymocytes. The DP cells cannot survive in vitro for retroviral manipulation, while the SP cells are untransducible. Therefore, this protocol can be applied to generate retroviral vector transducible DN thymocytes.

The OP9 bone marrow stromal cell line provides a useful in vitro system for the induction of lymphopoiesis from several sources of progenitors1. The first studies that used OP9 cells demonstrated that the lack of macrophage colony-stimulating factor (MCSF) expression contributed to the ability of the OP9 cell line to support hematopoiesis and B cell differentiation from bone marrow-derived hematopoietic stem cells (HSCs), as was later also shown for embryonic stem cells (ESCs)2,3,4,5. In previous studies, the generation of delta-like 1/4-expressing OP9 cells (OP9-DL1/OP9-DL4) enabled the induction of T cell lineage commitment6 and demonstrated the ability to successfully recapitulate thymic maturation7,8. Briefly, T cell development has been described by the sequential expression of CD4 and CD8 molecules. Immature thymocytes are &#34;double-negative&#34; (DN, CD4&#8722; CD8&#8722;) and can be further subdivided according to the surface expression of CD44 and CD25. DN thymocytes differentiate through the immature single-positive (ISP) stage, characterized by CD8 expression in mice and CD4 in humans, followed by the double-positive (DP) stage, characterized by the co-expression of CD4 and CD8, and, finally, the mature single-positive stage, characterized by the expression of CD4 or CD89. HSCs express the Notch1 receptor, which normally interacts with the delta-like 4 (DL4) expressed on thymic epithelial cells to induce T lineage differentiation10. Hence, interest in the OP9-DL1/4 model has progressively increased, leading to the extensive use of this approach in a wide variety of applications in the past two decades8,11,12,13. Although DL1 and DL4 are both able to support T cell differentiation in vitro, they show differential requirements in vivo, and a few studies have suggested that OP9_DL4 is more efficient than OP9_DL1 in recapitulating the mouse thymic environment7,14.
Among the potential applications of the OP9-DL1/4 system, there is particular interest in the combination of this system with the transduction of DN cells or HSCs with retroviral vectors. This combination is an effective way to manipulate gene expression during normal T cell development and leukemogenesis and has been shown to be an efficient method to induce or inhibit the function of a gene of interest15,16,17. This model has been particularly successfully used to study the collaboration between the oncogenes driving leukemia15 as it is flexible and enables the examination of the effects of multiple gene combinations in a reasonable time, in contrast to generating transgenic mice. Moreover, similar models have been used previously to assess the effects of introducing oncogenes into normal cells15,16,17. Additionally, retroviral transduction requires dividing cells for transgene integration18, and while lentiviral transduction would overcome this limitation by eliminating the need for dividing cells for transgene integration, we have been unable to achieve the successful transduction of DN thymocytes using lentiviral vectors. Thus, OP9-DL1/DL4 is a suitable tool for growing hematopoietic progenitor cells.
The standard protocol for the lymphopoiesis of transduced thymocytes on OP9-DL4 involves a series of coordinated steps that must be carefully performed to achieve a successful outcome. Although these are techniques that have been serving the community well for many years, often the protocols available in the literature are fragmented. As a result, each laboratory is forced to adapt and optimize different stages of the procedure, which can be time-consuming. Here, this protocol describes the isolation of DN thymocytes from the mouse thymus, followed by retroviral transduction and co-culture on OP9-DL4 stromal cells for further functional analysis. This established protocol has been shown to be efficient and reproducible in transducing primary thymocytes, followed by differentiation on OP9-DL4 cells or the induction of T cell acute lymphoblastic leukemia15.

<table><tbody><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>ACK Lysis buffer</td><td>Lonza</td><td>10-548E</td><td></td></tr><tr><td>BSA</td><td>Cell Signaling Technology</td><td>9998S</td><td></td></tr><tr><td>CD4 Microbeads</td><td>Miltenyi</td><td>130-049-201</td><td></td></tr><tr><td>CD8 Microbeads</td><td>Miltenyi</td><td>130-049-401</td><td></td></tr><tr><td>Centrifuge 5910R</td><td>eppendorf</td><td>5942IP802353</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Fetal calf serum HyClone FBS</td><td>ThermoScientific</td><td>&amp;nbsp;SH30910.03</td><td></td></tr><tr><td>LD columns</td><td>Miltenyi</td><td>130-042-901</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G7513</td><td>Freeze glutamine in aliquots and use freshly-thawed glutamine</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>P/N 52887</td><td></td></tr><tr><td>MEM-alpha Medium</td><td>Gibco</td><td>12561-072</td><td></td></tr><tr><td>OPTI-MEM I Reducing Serum Medium</td><td>Invitrogen</td><td>31985-062</td><td></td></tr><tr><td>PBS pH 7.2</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>pcL-Eco Plasmid</td><td>Addgene</td><td>12371</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pMIG Plasmid</td><td>Addgene</td><td>6492</td><td></td></tr><tr><td>Polybrene</td><td>Chemicon</td><td>TR-1003-G</td><td></td></tr><tr><td>Pre-Separation Filters</td><td>Miltenyi</td><td>130-041-407</td><td></td></tr><tr><td>recombinant hFLT-3L</td><td>PeproTech</td><td>300-19</td><td></td></tr><tr><td>recombinant IL-7</td><td>Peprotech</td><td>217-17</td><td></td></tr><tr><td>Retrovial packaging cell line Phoenix-Eco</td><td>Orbigen</td><td>RVC-10002</td><td></td></tr><tr><td>Syringe filter (0.45 &amp;micro;m)</td><td>Millipore</td><td>SLHV033RS</td><td></td></tr></tbody></table>

co-culture and transduction of murine thymocytes on delta-like 4-expressing stromal cells to study oncogenes in t-cell leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

The depletion efficiency can be assessed flow cytometrically by labeling the magnetically unlabeled cell fraction for CD4 and CD8 after immunomagnetic cell separation (MACS) and analyzing this on a two-dimensional bivariate dot-plot (Figure 3). A good yield of double negative (CD4&#8722;, CD8&#8722;) cells is 95% or above, as represented in Figure 3. Two of the most common causes of lower yield are the miscalculation of the microbeads based on the number of cells and the number of labeled cells exceeding the column capacity. It is recommended to&#160;choose the correct number of MACS columns according to the number of labeled cells.&#160;When working with thymocytes, the number of labeled cells (DP and SP cells) is almost equal to the number of total cells (more than 96%). Cell counting can be done in an automatic cell counter, a Neubauer chamber, or with any alternative method. As a result, the volumes allocated for cell counting may need to be adjusted depending on the specific machine and the chosen counting method.
It is important to determine the number of cells present before and after depletion. These cell counts are needed to calculate the number of LD depletion columns needed and to evenly distribute the DN thymocytes into the appropriate number of OP9-DL4 flasks. The controls for flow cytometry (unlabeled cells, cells labeled for CD4, and cells labeled for CD8) can be performed with the reserved pre-depletion sample, as this contains more cells. However, as most of the cells are expected to be retained within the column, the post-depletion sample to be labeled will require a higher volume. Consequently, adjustments may need to be made according to the labeling protocol selected.
When using vectors that express screenable markers, such as a fluorescence gene, the transfection and transduction can be roughly and empirically assessed by fluorescence microscopy (Figure 2). The transduction efficiency can be analyzed by harvesting the thymocyte from the OP9-DL4 monolayer, as described in step 8.3, and looking at the expression of a fluorescence gene by flow cytometry. The efficiency of transduction using an empty retroviral vector with GFP as the reporter gene was 84.2% (Figure 4).
T cell differentiation on OP9-DL4 cells can be observed 4 days after transduction. Flow cytometry is usually performed to assess the cell differentiation induced by the co-culture on OP9-DL4 cells and/or the transgene expression, as represented in Figure 5, where the cells were labeled for CD4, CD8, CD44, and CD25. There are several possible combinations of cell surface molecule labeling that have been shown to be useful for investigating the molecular and cellular mechanisms of T cell development in mice24,25,26,27. Therefore, the panels of fluorescent antibodies may vary according to the question of interest being addressed. The transduction of DN thymocytes with the empty retroviral vector pMIG, shown in panel B, presented approximately the same proportions of single positives (CD4+ or CD8+), double positives (CD4+/CD8+), double negatives (CD4&#8722;/CD8&#8722;), and its substages double-negative 1&#8211;4 (DN1&#8211;DN4) as the untransduced thymocytes, shown in panel A, indicating that T cell development was not affected by the transduction process.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64271/64271fig01.jpg" /> 
Figure 1: Diagram of the steps of thymocyte isolation, transduction, and co-culture. Abbreviation: DN = double-negative. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64271/64271fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64271/64271fig02.jpg" /> 
Figure 2: Co-culture fluorescence microscopy of GFP-transduced thymocytes or untransduced thymocytes and OP9-DL4 cells. (A) Untransduced thymocytes and (B) stable GFP-expressing murine thymocytes on OP9-DL4 at day 3 after the second transduction. An Olympus-IX71 fluorescence microscope with a 40x lens and a 480/30 filter suitable for GFP detection was used. Scale bar = 40 &#181;m. Abbreviations: GFP = green fluorescent protein; DN = double-negative. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64271/64271fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64271/64271fig03.jpg" /> 
Figure 3: Flow cytometry of thymocyte depletion. Representative flow cytometry plots analyzing the expression of CD4 and CD8 on thymocytes obtained from 7-8 week old C57BL/6J female mice (A) pre-depletion and (B) post-depletion of CD4+ and CD8+ using microbeads and LD columns according to the manufacturer&#39;s instructions. The dot plots on the left show gates based on the size and complexity of the event (FCS-A and SSC-A, respectively). The middle panel shows FSC-H versus FSC-A to gate single cells and exclude doublets. In the plots on the right, cells were defined as CD4 and CD8 from the single-cell gate. Abbreviations: FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; FITC = fluorescein isothiocyanate; PE = phycoerythrin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64271/64271fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64271/64271fig04.jpg" /> 
Figure 4: Flow cytometry of the thymocyte retroviral transduction efficiency. (A) Untransduced thymocytes co-cultured on OP9-DL4; (B) retrovirally transduced thymocytes on OP9-DL4 at day 3 post transduction. Abbreviations: FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; FITC = fluorescein isothiocyanate; PE = phycoerythrin; DN = double-negative. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64271/64271fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64271/64271fig05.jpg" /> 
Figure 5: Flowcytometry of transduced thymocytes after 4 days of OP9-DL4 co-culture. (A) Untransduced and (B) transduced with pMIG retrovirus. The cells were first gated on live cells and then gated based on size and complexity (FCS-A and SSC-A, respectively), followed by plotting FSC-H versus FSC-A to gate on single cells and exclude doublets. For untransduced cells, we used the following gate strategy. From the single-cell gate, the cells were defined as single positives (CD4+ or CD8+), double positives (CD4+/CD8+), and double negatives (CD4&#8722;/CD8&#8722;). Then, from the double negatives (CD4&#8722;/CD8&#8722;) gate, the cells were defined as CD44+, CD25+, CD44+/CD25+, and CD44&#8722;/CD25&#8722;, as shown in panel A. For the pMIG transduced cells, shown in panel B, from the single-cell gate, the cells were first defined as GFP+/GFP&#8722;, and then from GFP+ cells, the cell population distribution into the major T cell development stages, as defined by CD4, CD8, CD44, and CD25 expression, was determined using the same gate strategy as for the untraduced cells. Abbreviations: FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; FITC = fluorescein isothiocyanate; GFP = green fluorescent protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64271/64271fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia at JoVE.com

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

co-culture-transduction-murine-thymocytes-on-delta-like-4-expressing

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.0K Views.  National Institutes of Health. Het transduceren van onrijpe thymocyten met retrovirale vectoren en het kweken van deze cellen op OP9 DL4 stromale cellen is een uitdagende techniek. Dit gedetailleerde protocol bespaart onderzoekers tijd en moeite bij het optimaliseren van deze systemen. Deze techniek biedt een flexibel in vitro systeem voor het bestuderen van de effecten van genetische modificaties op onrijpe T-cellen en kan nuttig zijn voor het bestuderen van T-celontwikkeling en kanker.Gisele Rodrigues, een postdoc...

Video: Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Developmental Biology

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Immunology and Infection

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Medicine

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

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