Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Generation of HIS mice

1. Mouse husbandry of BALB/c –Rag2^null Il2RγC^null Sirpα^NOD (BRGS) mice
   NOTE: This strain is extremely immunodeficient, with no T, B, or natural killer (NK) cells. Therefore, rigorous measures must be used to prevent opportunistic infections. Maintain the colony on a diet containing trimethoprim and sulfadiazine on an alternating 2-week schedule with a normal diet. Maintain in the highest level of precaution housing room possible (e.g., a barrier shower-in facility with limited access).
   1. Maintain colonies of BALB/c –Rag2^null Il2RγC^null Sirpα^NOD (BRGS) and BALB/c Rag2^null Il2RγC^null Sirpa^Balb/c (BRG) homozygous mice as breeders.
   2. Breed BRGS^N/N×BRG^B/B to generate BRGS^B/N pups, to be used as recipients of human stem cells. In this colony, BRGS^B/N are healthier than BRGS^N/N, and engraft at equivalent levels (more than BRG).
2. CD34+ human stem cell isolation from umbilical cord blood (CB)
   NOTE: No antibiotics are used for this procedure. Therefore, good sterile technique is imperative.
   1. Place a 50 mL conical tube rack, as well as 3x 15 mL and ~10x 50 mL conical tubes in a sterilized biosafety cabinet (BSC). Spray a blood collection bag with 70% ethanol and let it dry in the BSC.
   2. Calculate the number of 50 mL conical tubes required for CB density gradient isolation = CB volume/15, rounded up and to an even tube number. Calculate blood volume per tube = CB volume/number of tubes. Pour blood carefully from the CB bag into each conical tube; this is a maximum of 15 mL per tube. Use an automatic pipettor and a 25 mL serological pipette to mix the blood 1:1 with sterile PBS by pipetting up and down.
   3. Use an automatic pipettor on low speed and a 10 mL serological pipette to slowly underlay the blood with room temperature (RT) 1.077 g/mL density gradient solution (see Table of Materials) without disturbing the interface. Keep the pipette tip from touching the tube bottom. Repeat for all tubes. Then centrifuge for 30 min at 850 x g, with no braking, at RT to ensure maintenance of the density gradient.
   4. Visualize the cellular buffy coat on top of the 1.077 g/mL density gradient as a cloudy white layer. Remove and discard the plasma layer down to about 10 mL above the buffy coat using a 25 mL serological pipette and an automatic pipettor.
   5. Collect the buffy coat with a sterile transfer or serological pipette. Use the pipette like a spatula to scrape the cells off the side of the conical tube while releasing the bulb (or pipetting slowly) to draw the cells up. Combine the buffy coats from two 50 mL conical tubes into one new 50 mL conical tube.
   6. Wash the cells by pouring 45 mL of sterile Hanks' balanced salt solution (HBSS) containing 2% fetal bovine serum (FBS) into each conical tube. Centrifuge for 11 min at 360 x g at RT.
   7. Aspirate the wash media down to the pellet in all tubes. Use a 10 mL serological pipette and an automatic pipettor to resuspend the first pellet in 10 mL of HBSS containing 2% FBS. Resuspend each pellet in the same 10 mL of HBSS, and rinse each tube with an additional 10 mL of HBSS to collect all the cells into a single tube.
   8. Pour 45 mL of sterile HBSS containing 2% FBS in the conical tube. Centrifuge for 10 min at 360 x g, at 4 °C.
   9. Aspirate the wash buffer down to the cell pellet and resuspend the pellet in 20 mL of magnetic cell separator buffer (see Table of Materials). Remove a small aliquot to count the cells with a hemacytometer at a 1:20 dilution in methylene blue. Add the number of blue and white cells. Centrifuge at 360 x g, for 10 min at 4 °C.
      NOTE: This protocol uses magnetic bead technology (see Table of Materials). The protocol can be modified for use with any cell-separation technology, with sufficient purity and yield of the CD34+ stem cells.
   10. Aspirate the supernatant and resuspend the CD34+ cell pellet isolated from cord blood in 300 µL of magnetic cell separator buffer per 1 x 10⁸ cells. Add 100 µL of Fc receptor (FcR) blocking reagent first, and then 100 µL of CD34+ magnetic beads per 1 x 10⁸ cells. Incubate at 4 °C for 30 min (no ice).
   11. Add 5 mL of magnetic cell separator buffer per 1 x 10⁸ cells and spin at 360 x g for 10 min at 4 °C. Repeat the wash step and resuspend the pellet in 500 µL of magnetic cell separator buffer per 1 x 10⁸ cells in a 15 mL conical tube labeled "unfractionated".
   12. Label two more 15 mL conical tubes "CD34-" and "CD34+". Place the three 15 mL conical tubes (unfractionated, CD34-, and CD34+) to slots A1, B1, and C1, respectively, on a cooling rack (see Table of Materials). Separate the cells using the two-column positive selection program on an automatic magnetic cell separator (see Table of Materials) in a BSC, according to the manufacturer's instrument instructions.
3. Expanding and freezing CD34+ human stem cells
   1. Aliquot 10 µL of the recovered CD34+ cell suspension (2 mL) on a hemocytometer slide and count the cells under 10x magnification. Calculate the total number of CD34+ cells by multiplying the cell count by 2 x 10⁴. Divide the total CD34+ cell number by 250,000 to calculate the number of vials to freeze (50,000 cells per mouse pup prior to in vitro expansion).
   2. Prepare CB medium Iscove's 10% fetal calf serum (FCS) (plus 1 mL extra for filtering loss), supplemented with 40 ng/mL stem cell factor, 20 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L), and 10 ng/mL interleukin-6 (IL-6), and pass through a 0.22 µm filter. Resuspend the CD34+ cells at 100,000 per mL of CB medium, and incubate at 37 °C. On day 3, add an equivalent volume of CB medium without cytokines to the flask containing the cells and the CB medium with cytokines.
      NOTE: Addition of these cytokines to the CB medium promotes survival and expansion of the CD34+ cells while preventing differentiation.
   3. Harvest the expanded CD34+ cells on day 5. Pipette the cell suspension up and down and collect in a 50 mL conical tube. Add enough CB medium to cover the bottom of the flask. Using a cell scraper, scrape the entire bottom of the flask. Collect all the media into the same 50 mL tube, and centrifuge at 360 x g for 11 min.
   4. Resuspend the cells in 2 mL of CB medium. Save the final drop from the pipette into a 96-well plate for counting. Dilute the cells 1:1 in trypan blue and add 10 µL to the hemacytometer, then count and average cells from four quadrants. Calculate the total number of CD34+ cells by multiplying the cell count by 4 x 10⁴, and record the viability.
   5. Make n+1 mL of freezing medium, where n is the number of freezing vials calculated in step 1.3.1. Prepare freezing medium by adding 10% (v/v) dimethyl sulfoxide (DMSO) to FBS and keep on ice. Label the cryovials with CB#, CD34+ d5, and date.
   6. Spin down the CD34+ cells at 360 x g for 10 min at 4 °C. Aspirate the medium down to the pellet and resuspend the cell pellet in freezing medium. Aliquot 1 mL of cell suspension to each vial and divide any remainder evenly between the vials. Add the vials to an isopropanol cell freezer chilled to 4 °C, place at -80 °C, and transfer to liquid nitrogen to store for >90 days.
4. Irradiation of mouse pups
   1. Collect the BRGS^B/N pups, 1-3 days after birth, into an autoclaved plastic box with padding. Add a small amount of bedding with the pups. Label the box with the cage number and the number of pups.
   2. Set up the irradiator (see Table of Materials) for a dose of 300 rad. Set the box of pups into the irradiator and expose them to 300 rad. Take the pups back to their cage, place them in a pile, and cover with bedding.
5. Pup injections and CD34+ cell preparation
   1. Begin CD34+ cell preparation ~3 h post irradiation. Warm 10 mL of CB media in a 50 mL conical tube. Perform all steps in a sterile BSC.
   2. Retrieve one vial of in vitro expanded and frozen CD34+ cells for every four to six pups to inject. Rapidly thaw at 55 °C, until just a small amount of ice is visible, and add the cells to the warmed CB medium (the vial should still be cool to the touch.) Use 1 mL of medium to rinse each vial and spin the cells at 360 x g for 12 min at 4 °C.
      ​NOTE: Rapid thawing at 55 °C was found to yield better cell viability (90%-95%) than thawing at 37 °C.
   3. Aspirate the medium carefully. Resuspend the (small) pellet in 2 mL of CB media, gently mix, and add ~30 µL of the cell suspension to a single well in a counting plate. Dilute 1:1 in trypan blue, then add 10 µL to a hemocytometer and count and average the cells from four quadrants.
   4. Calculate the total number of CD34+ cells by multiplying the cell count by 4 x 10⁴ and then record the viability. Spin at 360 x g for 12 min at 4 °C.
   5. Aspirate the medium carefully and resuspend the cell pellet in 100 µL of sterile PBS per n+1 pups to inject, resulting in 250,000-450,000 CD34+ cells per mouse. Place the conical tube on ice in a transport container and travel to the vivarium for pup injection.
   6. Bring a heat lamp, diapers, 1 mL syringe, 18 G needle, 30 G needle, and the CD34+ cell preparation in sterile containers to the vivarium BSC. Place a sterile diaper ~2 ft underneath the heat lamp. Retrieve the cage with the litter to be injected and place it in the BSC.
   7. Assemble the syringe with an 18 G beveled needle. Matching the angle of the conical tube with the bevel of the needle, gently mix and draw up the cell suspension. Place the pups on the diaper to warm (watch for overheating). Remove air from the syringe and replace the 18 G needle with the 30 G needle, and then carefully push the syringe until the cell suspension is just at the needle tip.
      NOTE: Alternatively, an insulin syringe can be used.
   8. Take one pup at a time to the edge of the diaper away from the heat lamp. Immobilize the pup on its side under the thumb and forefinger, allowing for a clear view of the face. Notice the vein across the cheek under where the ear will be. Insert the needle shallowly into the vein (IV) nearest to the eye, and slowly inject 50 µL of cells.
   9. Check if a bubble is forming from a subcutaneous injection. If so, insert the needle deeper and proceed to inject cells. A small drop of blood/hematoma will be visible when successful.
   10. With the pup still immobilized, perform an intrahepatic injection (IH) with another 50 µL of cells (100 µL total per pup of IV+IH). The liver can be visualized as a dark spot between the white milk band and the thoracic cage. Place the injected pup on a diaper further away from the un-injected pups and heat.
       NOTE: IV injection results in better long-term chimerism than IH injection alone, but IV injections are not always successful. Therefore, splitting the injection between IV and IH ensures engraftment into a larger percentage of mice.
   11. Repeat both facial vein and IH injections for all pups. Clean off any blood, return pups to the nest in their cage, and cover with bedding.