Ex vivo culture can hamper the repopulation potential of hematopoietic stem and progenitor cells. Our protocol preserves the stemness during the culture and thus improves the frequency of gene-modified cells in vivo. Hematopoietic stem cell gene therapy is currently being employed for monogenic diseases and infectious disease like HIV.
The HSPC gene therapy protocol heavily relies on ex vivo culture. Our protocol should be compatible with any procedure that employs ex vivo culture. Demonstrating this protocol will be Vigneshwaran Venkatesan, PhD student from my laboratory.
After isolating the HSPCs, culture them in stem cell culture media that contains cytokines and small molecules Resveratrol, UM729, and StemRegenin1. Before gene editing, set the ThermoMixer at 37 degrees Celsius to reconstitute the guide RNA, then preheat the TE buffer at 37 degrees Celsius for 10 minutes. Centrifuge the synthetic chemically-modified sgRNA vial at 11, 000 times g for 1 minute at 4 degrees Celsius and add 15 microliters of TE buffer to the vial containing 1.5 nanomolar of lyophilized sgRNA and mix by gentle swirling to get a final concentration of 100 picomolar per microliter.
Incubate the vial in the ThermoMixer with minimal shaking at 37 degrees Celsius for 30 to 40 seconds. Gently tap the vial on the sides and spin the vial to collect 15 microliters of sgRNA. Make 1-microliter aliquots and store them at minus 80 degrees Celsius for up to 1 year.
For nucleofection, pellet 2 times 10 to the 5th cells by centrifuging at 200 times g for 5 minutes at room temperature. Once the supernatant is discarded, resuspend the pellet in 20 microliters of the buffer. Gently mix the cell suspension with the prepared RNP complex avoiding air bubbles.
Now, transfer the suspension to the commercial nucleofection strip and electroporate the cells using the 4D-Nucleofector by selecting the pulse code DZ-100. To the electroporated cells in the nucleofection strip, add 100 microliters of pre-incubated culture media and leave the cells undisturbed for 10 minutes at room temperature. At the end of the incubation, transfer the contents to the culture plate as per the experimental requirements.
Place the NSG mice in the pie cages in 6 to 8 hours prior to HSPC transplantation. Irradiate the mice at 3.5 gray using a commercially-available irradiator. For NBSGW, pre-condition 6 to 8-week-old male and female mice by intraperitoneally injecting busulfan at a dose of 12.5 milligrams per kilogram of body weight 48 hours before HSPC transplantation.
For infusing 1 mouse, pellet 6 times 10 to the 5th cells in a 1.5-milliliter tube by centrifuging at 200 times g for 5 minutes. at room temperature. Gently pipette out the supernatant without disturbing the pellet and resuspend the cell pellet in 100 microliters of PBS.
Place the pre-conditioned NSG or NBSGW mice in the mouse restrainer and infuse the HSPCs by tail vein injection. Hold the mouse tail and gently push the plug to restrain the mouse. Gently wipe the mouse tail with 70%ethanol.
Using a 31 gauge insulin syringe, aspirate 100 microliters of the cell suspension. Now, direct the light from the infrared lamp onto the tail for 30 to 40 seconds, covering the body area of the mouse with folds of tissue paper. To maintain the tail in the planer axis with the syringe, lift it with the left index finger.
Gently insert the bevel part of the needle into the left or right caudal tail vein at an angle of 20 degrees. Push the plunger to infuse the cell suspension into the vein and apply gentle pressure near the pierced region with tissue paper and pull out the needle. After anesthetizing, position the animal in ventral recumbency and gently scruff the mice to open the eye, allowing the eye's globe to protrude slightly.
Gently insert the pasture pipette at a 30 to 45 degree angle into the medial canthus of the eye under the nictating membrane. After placing the Pasteur pipette at the proper position, apply slight pressure to the tube and begin to rotate the tube gently. After collecting 50 to 80 microliters of peripheral blood, gently withdraw the pipette from the medial canthus of the eye.
Post-euthanization, make a vertical incision 1 centimeter above the urethra and extend until 1 centimeter below the diaphragm. Cut horizontally at the incised area's corners to open the abdominal region. After dissecting the femur and tibia, use scissors to remove the soft tissues attached to the femur and tibia and gently scrub the bones with a tissue paper.
Use a scalpel to make a small hole with a diameter not more than 0.2 centimeters at the bottom of a 0.5-milliliter microcentrifuge tube. Remove the proximal ends of the bones using a scalpel and place the bones with the cut side facing toward the hole of the 0.5-milliliter micro centrifugation tube. Now, place the 0.5-milliliter tube with the bones in the 1.5-milliliter tube containing 100 microliters of sterile PBS.
Close the lid, spin the tubes at 200 times g for 3 minutes under sterile conditions at room temperature and discard the 0.5-milliliter tubes containing bones with an empty marrow cavity. To the 1.5-milliliter reaction tube containing bone marrow, add 1 milliliter of PBS, then using a 1-milliliter pipette, gently resuspend the cells 10 times. Transfer 1 milliliter of the cell suspension to a 15-milliliter tube containing 9 milliliters of RBC lysis buffer.
Incubate the cells in ice for 7 minutes by gently inverting the tube every two minutes. After incubation, centrifuge the tube at 200 times g for 5 minutes at room temperature and repeat the washing until a clear pale white pellet is observed. The frequency of live cells and CD34-positive, CD90-positive cells have increased in the RUS group after 48 hours of nucleofection.
After 72 hours, the percentage and frequency of indel increased in the RUS-treated group as compared to DMSO-treated group suggesting an increase in gene editing. The absolute number of CD34-positive, CD90-positive cells, and total nucleated cells are significantly higher 48 hours post-editing. Flow cytometry analysis of human CD45-positive cells in the NSG mice showed increased engraftment in the culture conditions.
Analysis of the gene-editing frequency in the mouse BM cells showed increased engraftment of gene-edited HSPCs in RUS-supplemented culture conditions. Culturing the HSPCs at a confluence of 200, 000 cells per mL is crucial to improve the functional stem cells. Amplifying the cord blood stem cells and the iSPC-derived stem cells are the new areas in which this protocol is being tested.
