Transduction of Primary AML Cells with Lentiviral Vector for In Vitro Study or In Vivo Engraftment

Summary We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary non-adherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).


SUMMARY
We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary nonadherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).

BEFORE YOU BEGIN
This protocol requires a concentrated stock of shRNA in lentivirus to infect the primary cells. A brief outline for the preparation of the concentrated virus is provided below. The reader is also referred to (Seneviratne et al., 2019) for more details. Proper institutional approvals for biosafety, use of animals, and human samples should be obtained before starting this protocol. This protocol provides a reliable transduction efficiency in hard-to-transduce primary AML cells with minimal toxicity. We have used this protocol to transduce 18 patient samples

STEP-BY-STEP METHOD DETAILS
Interaction of Primary AML Cells with Concentrated Virus (Day 1)

Timing: 6-7 h
In this section the virus will be spun down onto Retronectin-coated plates. The primary AML cells will be incubated with virus for transduction.
CRITICAL: Next steps should be performed in a containment lab equipped to handle viral samples.
1. Thaw the vials of concentrated virus in a 37 C water bath. 2. Add the 500 mL of concentrated virus onto Retronectin-coated plates (none in corner wells), spin plates at 3,000 rpm for 5 h at 4 C. Alternatives: This step can be performed at 20 C-22 C if preferred as no severe toxicity from the concentrated virus was observed using this temperature.
CRITICAL: After the addition of cytokines, the medium should not be heated more than 20 min in a 37 C water bath. In addition, the medium is photosensitive and should be covered with aluminum foil at all times.
Pause Point: Medium can be kept for~2 months at 4 C.
5. Thaw frozen primary AML patient sample in 37 C water bath (5-10 million cells). 6. Add thawed AML sample to 50 mL IMDM+10% FBS medium. Spin at 2,000 rpm for 10 min to pellet. Re-suspend pellet in 10 mL X-Vivo + BIT + GFs medium at a cell count of 5 3 10 5 cells/mL.
CRITICAL: Thaw the patient samples as fast as possible and add sample immediately to the medium. Otherwise, the cells will clump, and the sample will be lost.
7. Add 10 mL protamine sulfate (final concentration 5 mg/mL) to 10 mL of cells in medium. 8. Add 1 mL of AML cells (5 3 10 5 /mL) to all 24 wells (4 corner wells are for No Virus controls) and then spin plate at 1,300 rpm for 10 min to increase the interaction between the cells and lentiviral particles. The spin time can be increased depending on experimental needs. 9. Incubate AML patient sample cells with the virus for~24 h at 37 C in a 5% CO 2 incubator.
CRITICAL: Throughout the procedure use sterile filter tips and individually wrapped plastic pipettes.

Collection of Transduced Cells (Day 2)
Timing: 1-2 h In this section cells will be collected and transduction efficiency will be measured by calculating the percentage of GFP positive cells. The cells will be injected into the sub-lethally irradiated immune compromised mice.
10. Collect the cells from each well and transfer them to sterile 50 mL tubes.

OPEN ACCESS
CRITICAL: The medium in each well should be pipetted up and down several times before the cells are transferred to the tubes. If this step is not performed, the majority of cells will be lost.

No virus control cells and virus treated cells should be collected in separate tubes.
12. Spin transduced cells at 2,000 rpm for 10 min. Decant the supernatant into 10% bleach. 13. Re-suspend AML cells in fresh X-Vivo10 + BIT + GFs medium at a final count of 1 3 10 6 cells/mL, final volume depends on the total cell count. 14. Aliquot 1 mL of cells to a new 24-well plate. The user can set up the plate as desired. A suggestion is given in Figure 1. 15. Leave overnight at 37 C, 5% CO 2 incubator. 16. The percentage of GFP positive cells in total viable cell population (7AAD negative) can be measured in an aliquot of cells that were not injected into the mouse using a flow cytometer. These values are required to calculate engraftment potential at the end of in vivo experiments.
Alternatives: GFP levels can be measured at days 3 and 4 as well.
17. To confirm the target gene knockdown, sort GFP+ cells at day 2-3 post transduction and perform RT-PCR. 18. Infected cells can be maintained in culture for up to 1 week for downstream in-vitro experiments. 19. To engraft the marrow of immune deficient mice, inject 3 3 10 5 to 1 3 10 6 of AML cells into immune deficient mice. One possible mouse strain are sub-lethally irradiated (200 rad) immunodeficient NOD/SCID (6-10 weeks) mice, conditioned with intraperitoneal injection of antimouse CD122 antibody generated in house using the TM-beta1 Hybridoma. (200 mg/mouse) 24 h prior to injection of cells. The engraftment was evaluated using anti-human CD45 and CD33.

EXPECTED OUTCOMES
The above protocol will generate primary AML blasts with target gene knockdown in infected cells while preserving their stem cell properties. The transduction efficiency will be between 15% and 30% ( Figure 2 , Table 1). After transduction, mRNA target knockdown and changes in gene expression in sorted cells can be assessed by qRT-PCR. By using flow cytometry and gating on transduced cells, functional studies including cell viability, and engraftment into immune deficient mice in primary and secondary transplants can be measured (Figure 3).

LIMITATIONS
The average transduction efficiency is between 15% and 30% as measured by GFP positive cells. These values must be kept in mind for downstream assays. The proliferation of primary AML cells in culture is limited.

TROUBLESHOOTING Problem
Lower than expected transduction efficiency.

Potential Solution
The transduction efficiency can be increased by enhancing the interaction of AML cells and lentiviral particles, by one or all of the following steps: While coating the plates with Retronectin, make sure that the wells are evenly coated and the whole surface of the well is covered. While removing the Retronectin and blocking BSA solution, make sure wells are not scratched and the liquids are aspirated from the edges of the well.
The centrifugation of viral particles should be performed for a full 5 h, lower times may decrease the cell and virus interaction. Cells cannot be co-spun for such long duration as it will create viability issues. Ensure that the X-Vivo + BIT + GFs medium has been prepared and stored properly as the medium preserves the viability of the AML cells. Over time, or with improper handling, the medium can spoil. After thawing of the medium and addition of cytokines, the medium is stable at 4 C for 2 months. The medium is sensitive to heat and light. Prior to use it should not be warmed in a 37 C water bath for more than 20 min and tubes should always be covered with aluminum foil.

Problem
Clumping of primary AML cells during thawing.

Potential Solution
Always thaw the primary AML cells in a 37 C water bath. Work fast and as soon as the sample thaws transfer it to 50 mL of IMDM + 20% FCS. Always use 50 mL of medium, using lower volumes may increase clumping of cells. The number of cells thawed is potential issue.

Problem
Non-Engraftment of GFP+ cells.

Potential Solution
The handling of primary patient samples is very important. Repeated thawing and freezing could lead to depletion of stem cell population. In addition, the recipient mice should be of 6-10 weeks of age and should be kept in specific pathogen-free cages.

RESOURCE AVAILABILITY Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact: Aaron D Schimmer (aaron.schimmer@uhn.ca).

Materials Availability
This study did not generate any unique reagents

Data and Code Availability
This study did not generate/analyze datasets of code.