Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

This protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center.

NOTE: Obtain approval from the local IACUC before performing experiments on animals.

1. Processing of Umbilical Cord Blood and the Isolation of Human HSPCs

1. Perform all steps of the protocol under sterile conditions in laminar flow cabinets.
2. Take umbilical cord blood (CB) collected in heparinized tubes and make the volume up to 35 mL by adding phosphate-buffered saline (PBS). Layer the sample on top of the lymphocytes separation medium (LSM) as illustrated in Figure 1 and centrifuge the LSM with the layered CB at 400 x g for 35 min at 4 °C with no brakes.
   NOTE: Dilute the blood carefully and gently to avoid mixing at the interface. 
3. Remove the top LSM and plasma layer carefully and transfer the white buffy coat interface to a new tube using a transfer pipette.
4. Resuspend the buffy coat in 30 – 40 mL of ice-cold buffer (PBS + 0.5% bovine serum albumin [BSA] + 2 mM ethylenediaminetetraacetic acid [EDTA]). Using a pipette, combine 20 µL of the cell suspension with 20 µL of 0.4% trypan blue and pipette 10 µL of the mixture into the outer opening of either of the two chambers of a counting slide and insert the slide in an automated cell counter to count the cells.
   NOTE: Use ice-cold buffer in all steps, as it helps keep the cells viable.
5. Centrifuge the cells at 300 x g for 10 min at 4 °C and aspirate the supernatant carefully. Then, add 300 µL of ice-cold buffer.
6. Add 100 µL of human Fc receptor blocking reagent and 100 µL of monoclonal mouse anti-human CD34 antibody-conjugated microbeads for up to 1 x 10⁸ cells (see the Table of Materials). Incubate for 30 min at 4 °C, add 10 mL of ice-cold buffer to wash the cells, and centrifuge at 300 x g for 10 min at 4 °C.
   NOTE: Scale this up according to the cell number if more than 1 x 10⁸ cells are present.
7. Carefully remove the supernatant, resuspend the pellet in 500 µL of buffer, and proceed with the magnetic separation step to enrich HSPCs.
8. Place a positive selection LS column (see the Table of Materials) in the magnetic-activated cell sorting field and pass it through with 3 mL of buffer.
9. Load the sample to the LS column that can entrap microbeads bound to human CD34+ in samples and allow it to flow under the influence of gravity into the collection tube.
10. Wash the column 3x with buffer and collect the elute in the same collection tube of the CD34^– fraction of cells.
11. Plunge the column with 5 mL of buffer to elute the CD34⁺ cells into a new collection tube. Repeat the procedure to achieve a purity of >90%.
12. Count the eluted CD34⁺ cells using trypan blue dye in a hemocytometer. After counting, centrifuge the CD34⁺ cells at 300 x g for 5 min and discard the supernatant.
13. Resuspend the CD34⁺ cells in 25 µL of PBS for an injection to be used immediately in transplantation or cryopreserve the cells at a concentration of 1-2 million/mL in freezing medium (Roswell Park Memorial Institute medium [RPMI 1640 medium] + 50% fetal bovine serum (FBS) + 10% dimethyl sulfoxide [DMSO]) for further use in transplantation.
    NOTE: Always recount viable cells before using them in transplantation.
14. To check the purity of the CD34⁺ elute, take 50 µL of the suspension and incubate it with 10 µL of PE-conjugated anti-human CD34 antibody for 30 min at 4 °C. After the antibody incubation, wash the stained cells with PBS, resuspend them in 100 µL of PBS, and then, proceed to perform flow cytometry. Add an additional tube of cells with no antibody to design the gate in the flow cytometer.
15. After acquisition, analyze the data by selecting the region of interest on a forward scatter (FSC) and side scatter (SSC) plot, followed by gating for single cells on FSC-area and FSC-height plots. Gate CD34-positive cells on single cells in the PE channel and SSC-area plot.

2. Preparation of Human Hepatocytes for Transplantation

1. Remove the cryopreserved hepatocytes from the liquid nitrogen, quickly submerge the vial in the water bath, and thaw for approximately 90 – 120 s.
2. Remove the vial cap and pour the thawed hepatocytes into the 50 mL conical tube of the warmed thawing medium.
3. Suspend the cells by rocking the 50 mL tube by hand for a few seconds.
   NOTE: Do not vortex the tube.
4. Pellet the cells at 100 x g for 8 min at room temperature.Wash the pelleted cells in PBS with 0.1% BSA and pool them with either fresh or thawed HSPCs (ratio 10:1) in PBS in a final volume of 80 µL/mouse.

3. Animal Handling, Screening, Genotyping, and Treatment for Human HSPC and Hepatocyte Transplantation

1. Animal handling
   1. As a result of severe immunodeficiency, breed, house, and handle TK-NOG mice under aseptic conditions.
   2. Always wear a lab coat, gloves, shoe covers, and a face mask to prevent infection with potentially pathogenic microorganisms.
   3. Use sterile gloves and instruments for surgery and handle the animals aseptically throughout the surgery.
2. Selecting TK-NOG mice for the experiment
   1. Maintain the TK-NOG strain colony by breeding female TK-NOG mice with male non-TK-NOG littermates and select transgenic offspring by genotyping.
      NOTE: Perform genotyping (see step 3.3) to determine the presence or absence of the transgene in newborn male and female mice at the time of weaning.
   2. Select males at 6 – 8 weeks of age for transplantation due to their high sensitivity to the GCV-mediated depletion of HSV-TK transgene-expressing hepatocytes²¹.
   3. Ear-tag the mice at the time of weaning or surgery to ease identification. Note down the weight and health status of the animals.
3. Genotyping for the presence of the HSV-TK transgene using quantitative real-time polymerase chain reaction
   1. Perform genotyping at the time of weaning (usually at 3 – 4 weeks of age). For genotyping, cut a piece of the mouse ear in a laminar flow biological safety cabinet to maintain sterility and extract genomic DNA by using a genomic DNA isolation kit.
   2. Amplify genomic DNA extracted from ear piece in a 20 µL reaction mixture to screen for HSV-TK transgene under control of human albumin promoter by adding 1 µL of forward primer 5'-CCATGCACGTCTTTATCCTGG-3', 1 µL of reverse primer 5'-TAAGTTGCAGCAGGGCGTC-3', 0.5 µL of FAM probe 5′-FAM-AATCGCCCGCCGGCTGC-MGB-3', and 10 µL of master mix on a real-time polymerase chain reaction (PCR) instrument²².
   3. Set the real-time PCR settings as follows: 60 °C for 30 s (preread stage), 95 °C for 10 min (hold stage), 40 cycles of 95 °C for 15 s and 60 °C for 1 min (PCR stage), and 60 °C for 30 s (postread stage).
      NOTE: A cycle of threshold (Ct) below 22 is considered positive for HSV-TK transgene.
4. Treatment using ganciclovir and treosulfan
   1. Using 27 G needle, inject the TK-NOG mice with intraperitoneal GCV injections (6 mg/kg) 2x a day at day 7 and at day 5 in 100 µL of saline before surgery to deplete the mice's transgenic parenchymal cells (as shown in the experimental strategy in Figure 2)²³.
   2. On days 3, 2, and 1 before the surgery, precondition mice with nonmyeloablative intraperitoneal doses of treosulfan (1.5 g/kg/day) in 100 µL of saline, using a 27 G needle.
   3. One day before the surgery, draw two to three drops (~100 µL) of blood from the submandibular vein by pricking it with a 5 mm lancet, and isolate the serum by centrifuging (1,500 x g for 10 min at 4 °C) for the alanine aminotransferase (ALT) assay to assess the degree of liver damage.
5. Preparation for the surgery
   1. Use clippers to shave the mouse's fur surrounding the incision site (at the left of the peritoneal wall) before surgery.
   2. Adjust the oxygen flow to 1 L/min and the isoflurane flow to 3% – 5% in an induction chamber using a mouse anesthesia machine. Place one mouse at a time in the induction chamber for anesthesia.
   3. Attach the one end of a sterile extension tube (with a holding capacity of 550 µL of suspension; see the Table of Materials for specifications) to a 30 G needle and the other end to a 1 mL syringe.
   4. Fill the syringe with the suspension (80 µL/mouse) of pooled HEPs and HSPCs (see section 2), fit the syringe in the notch of a repetitive dispensing pipette, and adjust the dispenser to dispense 10 µL in each press.
   5. Once the mice are anesthetized (usually after 3 – 4 min), switch the isoflurane flow to the nose cone and reduce the isoflurane flow rate to 2% – 3%.
6. Intrasplenic transplantation of human HSPCs and hepatocytes in mice
   1. Perform all surgery steps in a laminar flow cabinet under sterile conditions.
   2. Place a clean sterile drape over the working surface and scrub the left side of the body of each mouse with 10 % povidone-iodine followed by 70 % isopropyl alcohol, before making an incision.
   3. Make a small incision (~1 – 1.5 cm in length and 5 mm deep) in the skin, muscle, and peritoneum at the left of the peritoneal wall with Vanna's type scissors to enter the peritoneal cavity approximately 5 mm below the lower edge of the rib cage.
   4. Locate the spleen, pull it slightly with forceps to the operating area for easy access, and insert the 30 G needle into the lower pole of the spleen.
   5. Unlock the plunger of the dispensing pipette and dispense 10 µL of the volume at a time, with a limit of 60 – 80 µL per spleen. Retract the needle slowly and clip the spleen with ligating clips using a ligation applier.
   6. Push the spleen back into the body cavity with cotton-tipped applicators wetted with sterile PBS. 
   7. Close the muscle layer of the abdominal wall using an absorbable suture interrupted suture pattern (not continuous). Accomplish skin closure with an interrupted suture pattern using non-absorbable sutures.
   8. Use warm water circulating pads to protect the mice from hypothermia after surgery.
   9. Remove non-absorbable sutures 10-14 days after surgery.
7. Postoperative care
   1. When the transplanted animal wakens, inject analgesic buprenorphine (0.1 mg/kg) intraperitoneally, 2x a day for a consecutive 3 days.
   2. Observe the animals at least 1x a day until they return to normal physical conditions.
      NOTE: Check each animal's body weight, since some mice may lose weight postsurgery. Mice typically regain their original weight in 1 to 2 weeks.

4. Engraftment Validation of the Human Liver by ELISA and the Human Immune System by Flow Cytometry

NOTE: Evaluate the reconstitution of the human liver and immune system monthly, starting 1 month posttransplantation by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively.

1. Collect blood samples from the submandibular vein using lancets in EDTA tubes, and centrifuge at 1,500 x g for 10 min at 4 °C. Isolate serum to check human albumin levels to assess the engraftment efficiency of the mouse liver for transplanted human hepatocytes, using a human albumin ELISA quantitation set (see the Table of Materials) by following the manufacturer's instructions.
   NOTE: Do not discard the pellet and use the pelleted cells for a flow cytometry analysis to evaluate the human immune system reconstitution.
2. Resuspend the cell pellet without serum in 35 µL of FACS buffer (PBS + 2% FBS) and stained with 5 µL of mouse-specific CD45 (concentration 0.5 mg/mL), 5 µL each of human-specific antibodies CD45 (0.1 mg/mL), CD3 (0.2 mg/mL), CD8 (0.1 mg/mL), and CD19 (0.5 mg/mL), and 20 µL of CD4 (0.25 mg/mL) and CD14 (0.25 mg/mL) each for 30 min at 4 °C, to check the development of a functional immune system from CD34⁺ HSPCs.
   NOTE: Consider adding one additional tube of unstained cells to determine the gating of the stained cells.
3. After incubation, transfer the stained suspension (~100 µL) in a polystyrene round-bottom flow cytometry tube and use 2 mL of 1x lysis buffer (see the Table of Materials) by diluting 1 part of 10x lysis buffer with 9 parts of distilled water and incubating 10 – 15 min to lyse red blood cells.
   NOTE: Observe turbidity to evaluate the red blood cell lysis. Once the sample becomes clear, the lysis is complete.
4. After the lysis, add 3 mL of the FACS buffer in the tube and centrifuge at 300 x g at 4 °C for 5 min to get a pellet. Repeat the washing by adding 3 mL of FACS buffer to the pellet and centrifuge at 300 x g at 4 °C for 5 min.
5. Fix the cells in freshly made 1% paraformaldehyde (PFA) and acquire stained cells on the flow cytometer, analyzed with flow software.
6. For the analysis, select lymphocytes gating on an FSC/SSC plot, followed by single-cell gating on FSC-area/FSC-height.
7. Further, gate for human-specific CD45 (hCD45) on the single-cell population and include mouse-specific CD45 (mCD45) for the exclusion of cells of murine origin. Strategize gating of the stained population based on the gating of unstained cells.
8. Gate hCD45⁺ cells to determine the frequency of CD3⁺ T cells and CD19⁺ B cells. Gate T cells to determine CD4 and CD8 subsets. To evaluate monocytes, gate on hCD45 to determine CD14⁺ monocytes.

5. HIV Infection of TK-NOG Mice and Its Effect on the Human Liver and Immune System

1. Handle the HIV-1 virus and all infected mice in a designated biosafety level 2 facility.
   CAUTION: Autoclave and discard all HIV-infected wastes in double biohazard bags. For safety reasons, wear cut-resistant gloves while handing HIV-infected mice.
2. Wear personal protection equipment (PPE), including a disposable coverall gown, shoe covers, a face mask, and double gloves at all times while working with the virus.
3. Select mice with a reconstitution of more than 15% of human CD45⁺ cells (tested in step 4.7) and with a presence of human albumin in the serum for HIV-1 infection (tested in step 4.1).
4. Inject the mice with 1 x 10³ to 1 x 10⁴ tissue culture infectious doses 50 (TCID₅₀) HIV-1_ADA in a volume of 100 – 200 µL per mouse, intraperitoneally.
5. Euthanize the HIV-infected mice 5 weeks postinfection by using isoflurane (with an isoflurane vapor concentration of >5%).
6. After euthanizing the mice, collect blood in EDTA tubes by a cardiac puncture for the isolation of serum, to see the effect of HIV-1 on the liver by evaluating human-specific albumin levels by ELISA (see step 4.1) and blood cells to check for changes in the human immune cells using flow cytometry (see steps 4.2 – 4.8).
   NOTE: Assess the peripheral viral load 5 weeks postinfection on a bioanalyzer to confirm if the mice are infected.
7. After drawing blood, excise the liver from the euthanized mice.
8. For the liver excision, expose the abdominal cavity by making an incision of 1.5 – 2 cm long and 0.5 cm deep in the skin, muscles, and peritoneum, from the xyphoid. Make a cut perpendicular to the spine between the liver and the diaphragm. Lift the liver and sever any membranes attaching it to the stomach and intestines.
9. Collect and fix the liver in 4% paraformaldehyde overnight and follow a standard immunohistochemical protocol to evaluate the effect of HIV on CK18⁺ hepatocytes by using human-specific CK18 antibody⁸.