ヒト肝幹細胞を用いたヒト化マウス肝臓の生成

The overall goal of this surgical stem cell transplant procedure is to generate a novel chimeric mouse model with human liver function using human immature hepatic stem progenitor cells that allow cell proliferation, maturation and differentiation for the acquisition of drug metabolism. This method provides a powerful tool as to create a micro-environment to support human donor hepatic stem cell progenesis to expand, differentiate and reconstitute the injured mouse recepient liver. The main advantage of this technique is that it matured proliferated cells can be used and that the liver almost completely repopulates.

To begin, make a one milligram per milliliter diphtheria toxin or DT stock solution by adding one milliliter of Phenolized 0.85%sodium chloride solution to one milligram of DT.Perform an intraperitoneal injection of DT by holding an albtrek scid mouse in dorsal recumbency and below the bend of the knees left or right of the midline, insert the needle at a 45 degree angle to the body. Using an insulin syringe, inject 100 microliters of freshly prepared 0.3 microgram per milliliter of DT per 20 gram mouse. At 48 hours post DT injection, place the mouse in a restrainer and warm the tale in a 30 degree Celsius water bath for approximately 10 minutes to dilate the blood vessels.

Then two centimeters from the tip of the tale, use a sharp scalpel blade to make a one millimeter nick in the tale and use a capillary tube to collect the blood. Centrifuge the microcapillary tube containing the blood at 1500 times G for five minutes and collect the serum by separating the supernatant from the cell pellet. Pipette 50 microliters of a one to 20 dilution of the serum onto GOT ASTP3 slides.

Then using a multi-purpose automatic dry chemistry analyzer according to the manufacturer's protocol, measure the absorbance of the reaction product at 650 nanometers. Using a cell sorter, isolate human hepatic stem cells from human primary fetal liver cells with the CDCP1, CD90 and CD66 cell antigens to obtain a CDCP1 positive, CD90 positive, CD66 negative subpopulation. Seed the cells onto collagen four coded culture dishes and incubate at 37 degrees Celsius.

When the cells reach 80 to 90%confluence, use PBS to wash the cells once. Then add 0.05%Trypsin EDTA and incubate at room temperature. When the cells appear to be floating or flowing freely under a microscope stop the enzymatic digestion by adding eight milliliters of DMF12 containing 10%FBS.

With a 10 milliliter serological pipette, slowly suspend the cells before transferring them to a 15 milliliter conical tube. Centrifuge the cells at 100 times G and four degrees Celsius for five minutes. Using 10 milliliters of DMF12 with 10%FBS, carefully resuspend the cell pellet and use a cell count instrument to determine the cell numbers.

Divide the cells into aliquots of one times 10 to the sixth cells per 50 microliters of PBS for each mouse and store on ice until transplantation. After anesthetizing a mouse according to the text protocol, check for a reflex by lightly pinching the rear foot pad. Then apply vet ointment to the eyes to prevent dryness.

With electric clippers, shave the surgical site and apply 70%ethanol and povidone-iodine to disinfect. Make a one centimeter skin incision in the left flank just below the costal border of the ribs. Then make an incision on the left abdominal wall followed by an incision on the peritoneum.

Next, carefully expose the spleen. Then at a five degree angle, insert a 100 microliter micro-injection syringe with a 32 gauge one half inch needle at a depth half the thickness of the spleen and from one end of the organ to the other directly inject one times 10 to the sixth human hepatic stem cells in 50 microliters of PBS in the organ and remove the needle. To prevent leakage of the cells following injection, use a finger to gently apply pressure for two minutes.

Place the spleen back into the mouse body and use a normal running suture for the musculature and skin to close the cavity. Immediately transfer the mouse on its side into a 37 degree Celsius pre-warmed cage. Avoid contact between the cage bedding and site of surgery and monitor the animal according to the text protocol.

At four to six weeks post transplantation, after euthanizing the mouse according to the text protocol, use surgical scissors to open the abdominal cavity by simultaneously cutting the cuteous and fascia. Using the scissors as a spreader, dissect the connective tissue above the peritoneum along the linea alba to open the peritoneal cavity. With forceps, lift the sternum.

Then puncture the diaphragm and cut through each side of the sternum up through the cervical girdle. Severe the vena cava on the thoracic side of the liver and pull the esophagus through the liver in the anterior direction. Remove the diaphragm and then using it as a handle, start pulling the liver out of the abdominal cavity.

The inferior vena cava will be holding the liver in place. Therefore, cut the inferior vena cava taking care not to prematurely liberate the right adrenal. Separate the lobes of the liver from each other at the junctions and embed them in OCT according to the manufacturer's protocol.

Freeze the OCT containing the liver tissue in the metal grids on the cryostat. With the cryostat, cut five micrometer thick sample sections at minus 18 degrees Celsius and mount them on room temperature microscope slides. Store the stock at minus 80 degrees Celsius.

Finally, prepare the slides for H&E staining according to previously reported procedures and ELISA according to the text protocol. As shown in these H&E stained sections, DT treated mice exhibited a disorganized hepatic architecture and displayed a correction for congestion with increased serum AST activity. At six weeks post transplantation, human hepatic stem cell derived cells reconstituted the liver structure by replacing the residential mouse hepatocytes.

Macroscopically, at four days post-transplantation, small human hepatic clusters that were uniformly distributed around the liver were detected. At 45 days, the clusters had proliferated into large clusters. As seen here, the presence of human hepatocytes in mouse livers was first confirmed at six weeks post-transplantation by staining with human nuclei and cytokeratin 8/18 antibodies.

Human ALB positive and CK19 negative hepatocytes resembled functional hepatocytes whereas cells that positively co-stained for human ALB and CK19 exhibited bi-potential capability for differentiating into hepatocytes and cholangiocytes. The presence of multiple round clusters surrounding the liver lobes with clear human nuclei and CK8/18 expression was detected demonstrating the colony forming capability of human hepatic stem cells in the humanized livers. Finally, the repopulation level reached up to 90%one month following transplantation and human Albumin secretion was detected in the repopulated mouse livers.

Once mastered, surgical cell transplantation can be done in 10 minutes if it is performed properly. While attempting this procedure, it's important to remember that liver injure should be restricted to supple lizer liver. Under cell leakage, cell transplantation should be prevented.

Combined with this procedure, other methods like gene transfection for inviro direct reprogramming with human cells can be performed in order to explore human cell convergence with our current model. After its development, this technique paved the way for researchers in the hepatology field to explore human drug metabolism, drug drug interaction, hepatic toxicity, and hepatisis virus in a humanized liver model. After watching this video, you should have a good understanding of how to isolate and expand the hepatic stem cell, induce liver injury, and transplant a cell via the spleen to generate a chimeric mouse model with human liver function.

Don't forget that diphtheria toxin can be controlled and important and precautions such as avoiding necrosis of the heart and the liver should always be taken while performing this procedure.