We thank Lucia Sammarco and her Lulu&#39;s Lemonade Stand for inspiration, motivation, and support. This work was supported in part by NIH R01 AI102893 and NCI R01 CA179363 (S.M.); NHLBI-HL087951 (S.R.); NIH-CA151893-K08 (M.J.R.); NCI 1R01CA164225 (L.W); the Alex Lemonade Stand Foundation (S.M.); the HRHM Program of the MACC Fund (S.M.; S.R.; M.S.T); the Nicholas Family Foundation (S.M.); the Gardetto Family (S.M.); the Hyundai Scholars Program (M.S.T.); Hyundai Hope on Wheels (S.R.); the MACC Fund (M.S.T. and S.M.); the Children&#39;s Research Institute, MCW (S.R.); and the Kathy Duffey Fogerty Award (M.J.R.).

All animal protocols followed the humane and ethical treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) within the Biomedical Research Center (BRC) of the Medical College of Wisconsin (MCW), Milwaukee, WI. The use of human peripheral blood mononuclear cells (PBMCs) was approved by the Institutional Review Board (IRB) of the Blood Research Institute of the Blood Center of Wisconsin, Milwaukee, WI.
1. Mice, cell lines, and vectors
<ol>
	<li>Obtain C57BL/6 mice from commercial vendors. Maintain the mouse colonies in pathogen-free conditions and use female and male mice between the ages of 6 and 12 weeks. Obtain de-identified human PBMCs from IRB-approved sources.</li>
	<li>Anesthetize the animals witha mixture of 20-30% v/v isoflurane in propylene glycol (1, 2-propanediol, USP grade) to anesthetize the mice. Apply vet ointment to the eyes to prevent dryness while the mice are under anesthesia.</li>
	<li>To euthanize the animals, perform cervical dislocation after the induction of anesthesia.
	<ol>
		<li>Restrain the mice by firmly grasping the base of the tail with one hand. Place a sturdy stick-type pen or the thumb and first finger of the other hand against the back of the neck, at the base of the skull.</li>
		<li>To produce the dislocation, push the hand restraining the head of the animal forward and push down while pulling backward with the hand holding the tail base. Verify the effectiveness of dislocation by feeling for a separation of cervical tissue.</li>
	</ol>
	</li>
	<li>Keep the animals in the chamber/cage for at least 5 min and remove them only when respiratory activity is absent or when there is a lack of a detectable heartbeat.</li>
	<li>Obtain K562 and YAC-1 cells from commercial vendors and maintain them in RPMI1640 medium containing 10% heat-inactivated FBS. Test these cell lines periodically to exclude the possibility of mycoplasma contamination.</li>
</ol>
2. Preparation and titration of lentiviral vectors
<ol>
	<li>Culture 5 &#215; 106 293T cells overnight in a T75 cm2 flask containing a solution of 20 mL of RPMI1640 medium with 10% FBS, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1 mM sodium pyruvate, 5% of 7.5% sodium bicarbonate solution, and 0.001% &#946;-mercaptoethanol. Place the flask in a 37 &#176;C incubator infused with 5.2% CO2.</li>
	<li>Harvest the 293T cells using trypsin (0.025%)/EDTA (1 mM) in phosphate-buffered saline (PBS). Add 5 mL of Trypsin/EDTA in PBS and incubate the flasks for 10 min in a 37 &#176;C an incubator to allow the detachment of 293T cells from the T75 cm2 flasks.
	<ol>
		<li>Collect the detached cells and wash them twice in PBS to remove any traces of trypsin and EDTA. Count the cells with a hemocytometer and adjust the cell number to one million cells per mL.</li>
	</ol>
	</li>
	<li>Transfect the 293T cells18 with 3.95 &#181;g of psPAX2, 1.32 &#181;g of pMD2G, and 5.26 &#181;g of pLEP-GFP-Puro (generated in-house at BRI by cloning EF1alpha promoter from pWPI in place of a CMV promoter in pLenti CMV-GFP-Puro)19.
	<ol>
		<li>Prepare 1 mL of 150 mM NaCl plus 63 &#181;L of 0.6 mg/mL polyethylenimine (PEI; 25,000 kD, linear). Mix well and then add the plasmids and mix. Incubate for 20 min at room temperature and then add it to cells.</li>
		<li>16 h post-transfection, replace the transfection medium with fresh medium plus 4.5 mM sodium butyrate. Harvest the supernatant containing virus 48 h post-transfection and concentrate by overnight centrifugation at 5,000 &#215; g. See the Materials Table.</li>
	</ol>
	</li>
	<li>Determine the viral titers using 293T cells by performing serial dilutions and assay by flow cytometry 72 h post-transduction20. Use the expression of green fluorescent protein (GFP) as a measure to quantify the viral titers.</li>
</ol>
3. Purification and expansion of murine primary NK cells
<ol>
	<li>Purify murine primary NK cells21. Briefly, pass single-cell spleen suspensions through nylon wool columns to deplete the adherent populations consisting of B cells and macrophages.</li>
	<li>Culture the non-adherent populations eluted from the nylon wool column that contains murine NK cells with 1,000 U/mL interleukin (IL)-2 in RPMI1640 medium with 10% FBS, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1 mM sodium pyruvate, 5% of 7.5% sodium bicarbonate solution, and 0.001% &#946;-mercaptoethanol (RPMI1640 complete medium).</li>
	<li>Change the medium from the flasks on day 4 of this culture to remove the non-adherent T and NKT cells; cells that remain adhered to the flasks are largely NK cells. Add 20 mL of fresh RPMI1640 complete medium and 1,000 U/mL IL-2 to replenish the flasks.</li>
	<li>Check the purity of the murine NK cell cultures on day 7 by flow cytometry with NK cell-specific markers20 using preparations with more than 95% of CD3-NK1.1+ population.</li>
</ol>
4. Purification and expansion of human primary NK cells
<ol>
	<li>Isolate PBMCs by density gradient from the buffy coats of healthy human volunteers. Briefly, carefully layer 35 mL of half-diluted (with HBSS) cells over 15 mL of density gradient in a 50-mL canonical tube. Centrifuge at 400 &#215; g for 30 min at 20 &#176;C in a swinging bucket rotor without brake.</li>
	<li>Use the commercial antibody-based negative selection kits to purify human primary NK cells according to the manufacturer&#39;s protocol.</li>
	<li>Following the isolation, determine the purity of the NK cell preparations by immunofluorescence analyses. Stain NK cell preparations with 2 &#181;g/mL anti-CD3 and 2 &#181;g/mL anti-CD56 antibodies at 4 &#176;C for 20 min. Wash the cells twice with PBS NK cell populations determined by the absence of CD3 and the presence of CD56 on the cell surface.</li>
	<li>Incubate the cell preparations with antibodies for 20 min at 4 &#176;C, wash with PBS, and analyze in a flow cytometer. Use NK cell preparations with more than 85% purity for the gene transduction assays20.</li>
</ol>
5. Transduction of murine and human primary NK cells with lentivirus
<ol>
	<li>Suspend mouse or human primary NK cells in a 24-well plate at 0.5 &#215; 105/mL of medium in the presence of GFP lentivirus supernatant at 5, 10, and 20 multiplicity of infection (MOI) and in the presence of Pb (8 &#181;g/mL), PS (8 &#181;g/mL) or dextran (8 &#181;g/mL).</li>
	<li>Centrifuge the plates at 1,000 &#215; g for 60 min.</li>
	<li>Without decanting the supernatant, culture the cells overnight (16-18 h) in a 37 &#176;C incubator infused with 5.2% CO2. </li>
	<li>Wash with 10 mL of PBS and resuspend in 2 mL of complete RPMI1640 culture medium in the presence of IL-2 (300 U/mL).</li>
	<li>Test the human and mouse primary NK cell-mediated cytotoxicity against K562 and YAC-1, respectively by performing 51Chromium (Cr)-release assays at varied effector-to-target-cell ratios22.
	<ol>
		<li>Briefly, give one million target cells 50 &#181;Ci of radiolabeled sodium chromate 51Cr. During the 4-h incubation time, they uptake the 51Cr into cellular proteins. At the end of the incubation, wash the cells to remove any unincorporated label.</li>
		<li>Calculate specific tumor cell lysis by the amount of absolute, spontaneous, and experimental release of 51Cr from the target cells. 
		NOTE: The calculation of the percentage of specific lysis from pentaplicate experiments was done using the following equation: % specific lysis = ((51Cr mean experimental release - 51Cr mean spontaneous release) / (51Cr mean maximal release - 51Cr mean spontaneous release)) &#1093; 100, where &#34;51Cr mean spontaneous release&#34; is the 51Cr released from target cells in the absence of NK cells and &#34;51Cr mean maximal release&#34; is the 51Cr released from target cells upon lysis by 2 N hydrochloric acid (HCl).</li>
	</ol>
	</li>
	<li>Harvest the transduced NK cells on day 7 by gently tapping the flasks.
	<ol>
		<li>Activate the harvested cells with titrated concentrations of plate-bound anti-NKG2D (A10) mAb by coating 96-well, highly protein-absorbent polystyrene plates with 2.5 &#181;g/mL concentrations of anti-NKG2D mAb overnight.</li>
		<li>Wash each well with 100 &#181;L of PBS three times before the addition of primary NK cells.</li>
	</ol>
	</li>
	<li>Collect culture supernatants using a multi-channel pipette between 16 and 18 h post-activation to quantify cytokines such as IFN-&#947;. Generate standard curves using the recombinant cytokines provided with enzyme-linked immunosorbent assay (ELISA) kits.</li>
	<li>Test the NK cell viability using annexin-V/7-amino-actinomycin D (7-AAD) staining23 and determine the percent of necrotic cells among the transformed NK cells using a flow cytometer.
	<ol>
		<li>To perform this assay, harvest the murine and human NK cells four days after transduction, wash two times with 10 mL of cold PBS at 500 &#215; g for 5 min each, and incubate with an Annexin-V (PE)/7-AAD kit.</li>
	</ol>
	</li>
	<li>Use appropriate flow cytometry software to analyze the data. Select the live-cell population and analyze the expression of GFP (FITC channel) as a measure of viral transduction24.</li>
</ol>

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The authors claim no financial conflict of interest.

This study demonstrates that use of dextran as a cationic polymer agent enhances the lentiviral transduction efficiency of both murine and human primary NK cells. Additionally, other cationic agents, such as Pb or PS, have no discernible effect on the delivery of viral vectors into human primary NK cells. Previously, it has been demonstrated that Pb can augment gene transduction in human T cells17. These results, however, suggest that neither Pb nor PS have a similar efficiency on human primary NK...

Natural killer (NK) cells are the major lymphocytic population of the innate immune system1. NK cells function as the first-line defenders of the host immune response against tumors and infections2,3,4. NK cells also play a central role in the development of tolerance through the secretion of potent cytokines and chemokines5. Due to their potent ability to target and eliminate tumor cells, multiple clinical trials are being conducted to evaluate donor-derived human NK cells as an adoptive immunotherapy for cancer6,7. In contrast to T cells, the developmental biology of NK cells has yet to be well-characterized8. This lack of knowledge is partially due to the absence of efficient techniques that deliver genes of interest to mouse or human primary NK cells. For these reasons, most NK-cell studies have been conducted in cell lines, rather than in primary cells. Therefore, the need for a reliable and efficient protocol to transduce primary NK cells with genes of interest is crucial.
The overall goal of this study was to formulate a consistent and reliable method by which primary human or murine NK cells could be transduced with lenti- or retroviruses.
Earlier studies that attempted to address this problem have been performed, largely using the transient transformation of primary NK cells. This includes plasmid transfection9,10, Epstein-Barr Virus (EBV)/retroviral hybrid vector11, vaccinia vectors12,13, and Ad5/F35 chimeric adenoviral vectors14. Despite the modest efficiency of these techniques, the transient nature of transduction makes them unsuitable for the long-term utilization of the genetically modified NK cells. A few recent studies have used retroviral vectors to transduce NK cells, requiring multiple cycles of infection to achieve an acceptable level of gene expression11,15. In contrast to retroviral vectors, lentiviral vectors can use host-cell nuclear import machinery to translocate the viral pre-integration complex into the nucleus. This is a major limiting factor in the replication of the virus in non-dividing cells, which include primary NK cells.
Interactions between different cell-surface receptors and viral particles permit viral uptake into the cell. The initial engagements between the viral envelope proteins and their cognate host receptors could be limited because of the potential negative charges existing between these two. The rationale behind many transduction techniques is that the addition of cationic polymers, such as polybrene (Pb), protamine sulfate (PS), or dextran, could give a positive charge to the cell-surface receptors and thereby augment the binding of viral envelope proteins. This will increase the fusion efficiency and the uptake of the viral particles by the cells16. Although it has been reported that Pb or PS can improve gene transfer in T cells17, their application did not have any effect in the transduction efficiency of primary NK cells. Moreover, a comparative analysis between these reagents using primary NK cells has not been performed. In this study, the transduction efficiencies of the three cationic polymers were compared. The results show that, among these three cationic polymers, only dextran significantly enhances efficient viral transduction into both mouse and human primary NK cells.

<table><tbody><tr><td>Dextran</td><td>Sigma-Aldrich</td><td>90-64-91-9</td><td></td></tr><tr><td>polybrene (Pb)</td><td>Sigma-Aldrich</td><td>TR-1003</td><td></td></tr><tr><td>protamine sulfate (PS)</td><td>Sigma-Aldrich</td><td>p3369</td><td></td></tr><tr><td>Trypsin</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>RPMI1640</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATALANTA</td><td>S11150</td><td></td></tr><tr><td>Penicillin</td><td>Corning</td><td>30-001-CI</td><td></td></tr><tr><td>B-mercaptoethanol</td><td>SIGMA</td><td>M3148</td><td></td></tr><tr><td>sodium pyruvate</td><td>Corning</td><td>MT25000CI</td><td></td></tr><tr><td>Interferon gamma (IFN-&amp;gamma; )</td><td>eBioscience</td><td>14-7311-85</td><td></td></tr><tr><td>Propidium lodding staining solution</td><td>BD</td><td>51-66211E</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher</td><td>L3000015</td><td></td></tr><tr><td>Isoflurane</td><td>PHOENIX</td><td>NDC 57319-559-05</td><td></td></tr><tr><td>NK cell negative selection kit</td><td>Stem Cell</td><td>19855</td><td></td></tr><tr><td>Yac-1</td><td>ATCC</td><td>TIB-160</td><td></td></tr><tr><td>K562</td><td>ATCC</td><td>CCL-243</td><td></td></tr><tr><td>Mice</td><td>Jakson</td><td>664</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>T75 flasks</td><td>Cornnig</td><td>430641U</td><td></td></tr><tr><td>antibody-based negative selection kits</td><td>Stem Cell</td><td>19055</td><td></td></tr><tr><td>&lt;sup&gt;51&lt;/sup&gt;Chromium (Cr)-release assays</td><td>perkin elmer&#039;s</td><td>NEZ030</td><td></td></tr><tr><td>ELISA kits</td><td>Ebioscience</td><td>00-4201-56</td><td></td></tr><tr><td>Sodium Butyrate</td><td>Sigma</td><td>5887-5G</td><td></td></tr><tr><td>Linear polyethylenimine</td><td>polysciences</td><td>23966-2</td><td></td></tr><tr><td>Ficoll</td><td>GE Life Science</td><td>17-1440-03</td><td></td></tr><tr><td>HBSS</td><td>Corning</td><td>21-022-CV</td><td></td></tr></tbody></table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

Dextran induces the efficient gene transfer of lentiviral vector in primary human and murine NK cells
Human NK cells were isolated and purified from PBMC (with a purity of more than 85%) and incubated overnight with rIL-2 300 U/mL. These primary NK cells were then transduced with GFP lentivirus at varied multiplicities of infection (MOI; 3, 10, and 20 IU per cell) in 24-well plates in the presence of 8 &#181;g/mL Pb, PS, or dextran...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

dextran-enhances-lentiviral-transduction-efficiency-murine-human

Research

JoVE Journal

Immunology and Infection

11.0K Views.  The Blood Center of Wisconsin. Het doel van deze studie was om formuleren van technologieën die het mogelijk voor succesvolle gene signaaltransductie in primaire natural killer (NK) cellen maken. De dextran-gemedieerde lentivirale transductie van mens of muis primaire NK cellen leidt tot hogere rendementen van gen expressie. Deze methode van gene transductie zal sterk verbeteren NK cel genetische manipulatie.

Video: Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Genetics

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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.

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 ...

Cancer Research

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

Developmental Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Biology

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

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