This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.
This method enables the generation of large numbers of human liver bud progenitors and hepatocyte-like cells with high purity. The ability to generate these liver cells could be important for regenerative medicine and other fields. By controlling the developmental signaling pathways to promote liver differentiation and suppress the formation of unwanted cell types, this method enables efficient generation of human liver bud progenitors and hepatocyte-like cells by six and 18 days of differentiation respectively.
The main advantage of this technique is the high purity of the human liver cells generated. To being this procedure, add 50 milliliters of IMDM to a conical flask containing a stir bar. Heat the medium to 50 degrees Celsius and add 0.5 grams of PVA while continuously stirring to prepare a PVA stock at a concentration of 10 milligrams per milliliter.
After this, remove the PVA solution from the heating pad and allow it to cool down to room temperature. Once the solution is cooled, filter it through a sterile, 0.2 micrometer filter. Prepare the CDM2 and CDM3 by combining the filtered PVA with other reagents as outlined in table one of the text protocol.
Use a sterile, 0.2 micrometer filter to filter all media. Then, prepare the remaining base media, CDM4 and CDM5, as outlined in table one of the text protocol. To prepare the differentiation medium, first thaw the frozen small molecules and/or growth factors at room temperature.
Aliquot out the required amount of base medium and add the specified small molecules and growth factors to the base medium at the appropriate concentrations. The day before use, thaw the matrix at four degrees Celsius. The next day, dilute the matrix at a ratio of one to 100 by adding 500 microliters of matrix into 50 milliliters of cold DMEM.
Pipette the diluted matrix into the required number of wells using just enough volume of solution to cover the surface of the well. Transfer the matrix-coated plate to an incubator at 37 degrees Celsius for at least 60 minutes. Immediately prior to seeding, aspirate the remaining matrix solution from the coated wells.
Then, aspirate the medium from a largely confluent hPSC culture plate and add commercially-bought dissociation agent, making sure to use just enough to cover the surface on which the cells are growing. Incubate the hPSCs in the dissociation agent at 37 degrees Celsius for five minutes or until some colonies begin detaching. Next, add DMEM and F-12 to dilute the dissociation agent.
Use a five milliliter serological pipette to gently pipette up and down multiple times to wash off all cells from the surface of the well. Collect the resuspended single cells in a 50 milliliter conical tube and dilute with DMEM and F-12 as needed. Centrifuge this tube of collected hPSCs at 350 times g and at four degrees Celsius for three minutes to pellet the cells.
While waiting for the centrifugation, aspirate the matrix from the plate in which the hPSCs will be seeded. Add a sufficient amount of mTeSR media to recipient wells to cover them. When the centrifugation is complete, carefully aspirate the supernatant, leaving the pelleted hPSCs at the bottom of the conical tube.
Resuspend the cell pellet in mTeSR supplemented with one micromolar of commercially-obtained Thiazovivin. Using a P1000 pipette, gently triturate two to three times to evenly resuspend the cell pellet into a single cell suspension. Immediately pipette 10 microliters of the cell suspension into a hemocytometer and count the number of cells.
Adjust the volume of the resuspended hPSCs with Thiazovivin-supplemented mTeSR to achieve the desired cell concentration for plating. Then, shake the plate in a cross pattern several times to make sure that the cells are evenly distributed. After the hPSCs have been plated for at least 24 hours, use a phase contrast microscope to check the morphology of the cells with specific emphasis on the diameter of plated hPSC colonies.
Next, prepare day one APS differentiation medium as outlined in table two of the text protocol. One day after seeding, aspirate the Thiazovivin-supplemented mTeSR from the plated hPSCs and briefly wash them with IMDM media. After this, add day one medium to the hPSCs.
Record the time and place the cells back into the incubator at 37 degrees Celsius. Continue with the subsequent differentiation steps by preparing the needed differentiation medium and adding it to the cells on the respective days of differentiation around the same time of day. In this study, enriched populations of liver bud progenitors and subsequently hepatocyte-like cells are generated from hPSCs.
By day two of differentiation, primitive streak cells have differentiated into day two definitive endoderm cells. The vast majority of these cells express SOX17 and FOXA2. By day three of differentiation, the endoderm cells have differentiated into foregut progenitors that appear polygonal in shape.
Later, by day six, the foregut progenitors have differentiated into liver bud progenitors. These liver bud progenitors express AFP, TPX3, and HNF4alpha. Across three hPSC lines, this method generates day six AFP-positive liver progenitors at an efficiency of approximately 89%Finally, after 18 days of liver differentiation from hPSCs, albumin-positive hepatocyte-like cells appear.
Morphologically, these cells appear epithelial, forming bright borders reminiscent of bile canaliculi. At this stage, the cytoplasm of the day 18 hPSC-derived hepatocytes appears darker than the nucleus. When seeding human pluripotent stem cells for differentiation, it is imperative to seed them at the right density and also to distribute them across the well by shaking the plate.
The ability to produce human hepatocytes in vitro is an enabling technology that will allow scientists to investigate mechanisms underlying liver development. This method produces large numbers of human hepatocytes in vitro which could be used to investigate liver function and disease and to eventually enable new therapies for liver failure.
