This method have answered key questions in the field, such as how to generate a human liver chimeric mouse model, familial hypercholesterolemia. The main advantage of this technique is that it allows performing drug tests in in vivo. This technique has syndication for the treatments of familial hypercholesterolemia.
As a new therapy for this disease, it can be tested using such chimeric animal models. Though this method can provide insight into familial hypercholesterolemia, it can also be applied to other inherited liver diseases. 24 hours prior to engraftment, thaw 40 microliters of extracellular matrix per mouse, by placing in an icebox, in a cold room.
Chill an insulin syringe for each mouse to be injected and a box of 200 microliter tips in a four degree Celsius refrigerator. One hour prior to engraftment, warm the cell dissociation enzyme, supplemented with 50 micrograms per milliliter of DNAase 1, to room temperature and place RPMI 1640 medium, supplemented with 20 percent serum replacement on ice. Take phase contrast images of iHeps to record their status, including cell morphology, growth and cell density.
Next, wash each well of iHeps with 2 milliliters of room temperature calcium and magnesium ion-free PBS twice and then add one milliliter of the pre-warmed cell dissociation enzyme to each well. Return the cells to the incubator for eight to ten minutes. Monitor the cell morphology under the microscope.
When most of the cells become round, add in equal volume of cold medium per well, pipette the cells gently to detach from the plate, and transfer the cell suspension to a new 15 milliliter tube. This step is critical for the hepatocytes reliability. If the cells are difficult to detach from the plate, some can be left on the plate.
And if the cells detach, Then pipette gently after centrifugation, to obtain a single-cell suspension. Repeat the cell-detachment procedure for each well until nearly all attached cells are collected. Then centrifuge at 200 g for three minutes at four degrees celsius.
Following centrifugation, remove the supernatant and re-suspend the cells with two milliliters of cold PBS in the fifteen milliliter tube. Pipette the cells gently to obtain a single-cell suspension. Then, pass the cells through a 40 micron cell strainer to remove aggregates.
Normally, one to two million cells about can be harvested after few-ter-ing. Next, add microliters of 0.4 percent Trypan blue solution to 20 microliters of cell suspension. Count the cells and record the concentration of cell suspension as C.Calculate the required volume of cell suspension to inject one million cells per mouse.
Then allocate the required volume of cell suspension into 15 milliliter tubes and centrifuge at 200 g for three minutes at four degrees Celsius. After removing the supernatant, re-suspend the cells in 27.5 microliters of cold PBS per mouse. And then add an equal volume of extracellular matrix for a final volume of 55 microliters per mouse.
Now, place one of the cold insulin syringes on ice. Unplug the piston, transfer 55 microliters of cell suspension into the syringe and then put the piston back. Discharge bubbles carefully, and put the syringe back on ice.
Ensure that LRG mice are properly anesthetized before beginning the injection procedure. Apply veterinary ointment over the eyes to prevent dryness during the anesthesia procedure. Once the mouse has lost muscle reflex to stimulation, place it in the right, lateral, decubitus position.
Apply a thick layer of depilatory cream to the incision area in the left flank, and leave for five to eight minutes. Remove the depilatory cream and hair by wiping the area with a water-moistened gauze pad. Scrub the left flank with 70 percent ethanol three times.
Then locate the spleen, which can be seen in the left flank. Followed by a final soaking with betadine for disinfection. After using scissors to make a 0.5 to one centimeter incision in the skin and abdominal wall, use forceps to exteriorize the spleen by gently pulling the surrounding adipose tissue.
Gently stabilize the spleen using a swab. Insert the needle of the insulin syringe three to four millimeters into the parenchyma of the spleen, and gently inject approximately 50 microliters of cell suspension. Retract the needle and place a cotton swab over the injection for one minute to prevent bleeding and spillage of material.
Return the spleen to the peritoneum, and close the wound with five-oh nylon sutures. After suturing, place the mouse in a clean cage with easy access to food and water. Begin by removing the internal organs from the freshly euthanized mouse so that the descending aorta, parallel to the spine, is visible.
Then dissect away the adjacent tissue, and remove the heart. Use fine scissors to dissect the aortae, placing each one into cold, oxygenated Krebs solution. After collection, transfer the aortae in Krebs solution to a silicone-coated Petri dish.
Pin the connective tissue to fix the aorta position without stretching it. Under a sterile microscope, use fine forceps and spring scissors to dissect the aorta free from the surrounding fat and adventitial tissue without damaging the vessel wall. Then cut each aorta into one and a half to two millimeter length segments.
Next, cut a two centimeter long piece of 40 micron thick stainless wire, and gently insert into the aorta lumen. Hold the wire to transfer the segment to the wire myograph chamber, filled with oxygenated Krebs solution. To measure the length of the aortae segments when studying contractility, place each segment perpendicularly in between the jaws, and record the D1 reading on the micrometer.
Then remove the segment and move the jaws together. Record the D0 reading and calculate the length of the segment. Next, clamp the wire and secure it with the screwdriver whilst placing the unstretched segment in between the jaws.
For normalization before the experiment, set the myograph to zero in the unstretched position. Then slowly move the jaws apart and observe the aorta tension change until reaching three millinewton. After 15 minutes, drain the solution from the myograph chamber and replace with fresh Krebs solution.
After waiting for another 15 minutes, again adjust the tension to three millinewton. Then change the standard Krebs solution to Krebs solution supplemented with 60 millimolar potassium chloride, to induce contraction for at least fifteen minutes. Rinse with fresh Krebs solution three times, then add increasing concentrations of phenylephrine.
For example, 10 nanomolar to 100 micromolar. Then, after washing out with standard Krebs solution, add a single concentration of phenylephrine, at about 70 percent of maximal contraction. When the contraction is stable, add increasing concentrations of acetylcholine at two-minute intervals.
For example, one to three nanomolar to ten to thirty micromolar to induce vasodilation. This image shows a representative whole-section scanned image of human albumin staining in a mouse liver repopulated with low-density lipoprotein receptor-deficient iHeps. Arrows indicate clusters of human iHeps engrafted into the mouse liver.
The clusters of engrafted human iHeps are seen in more detail here. This scatterplot graph shows the percentage of repopulated human albumin positive cells corresponding to iHep containing areas in the mouse liver, from different donor iPSCs. This is a representative image of immunohistochemical staining for human nuclei in a mouse liver, with engrafted familial hypercholesterolemia iHeps.
The bar graph shows the percentage of repopulated human nuclei positive iHeps from different donor iPSCs in LRG mouse livers. These are representative images of human albumin and human nuclei staining on two consecutive sections of a mouse liver, repopulated with wild-type iHeps. Finally, this image shows endothelium-dependent vasodilation of the aorta in familial hypercholesterolemia human liver chimeric mice, in response to increasing concentrations of acetylcholine.
After watching this video, you should have a good understanding of how to generate human liver chimeric mice, using human iPSC-derived hepatocytes.
