Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

This protocol provides a stem cell derived hepatic differentiation system that offers a reproducible tool for studying liver biology for both basic and clinical research. By combining a standardized and easy to follow protocol with off the shelf culture reagents, this system allows for the production of hepatic progenitors and hepatocyte like cells at a large scale. Maintain human pluripotent cells at 37 degrees Celsius and 5%carbon dioxide in a 10 centimeter dish on laminin-531, feeding them daily with 10 milliliters of stem cell maintenance medium per dish.

To prepare laminin 521 plates, dilute thawed laminin 521 in ice cold DPBS with calcium and magnesium to a final concentration of eight micrograms per milliliter. Add 250 microliters of the laminin solution to each well of 24-well plate or 50 microliters to each well of a 96-well plate. Gently rock the plate from side to side to evenly coat the wells, then seal the plates with a semi-transparent flexible film and store them at four degrees Celsius overnight.

On the day of cell seeding, warm the pre-coated plates in a cell culture incubator for 30 to 60 minutes. Aspirate the laminin 521 solution and add stem cell maintenance medium supplemented with 10 micromolar ROCK inhibitor to each well, then return the plate to the incubator. When cell count fluency reaches 70 to 80%aspirate the spent medium from the culture dish and wash each culture dish with five milliliters of DPBS without calcium or magnesium at room temperature.

Aspirate the DPBS wash from the culture dish, then add five milliliters of enzyme-free dissociation reagent to the dish and incubate the plate at 37 degrees Celsius for eight to 10 minutes until cells visibly detached from the dish. Gently detach the cells from the culture dish with a cell scraper, then pipette the contents of each well up and down with a five milliliter serological pipette to yield a single cell suspension. For each cell line, pool cells from all maintenance wells into a sterile 50 milliliter tube.

Wash each emptied culture dish with five milliliters of these stem cells maintenance medium and add the washes to the corresponding tube with the pooled cells. Perform three viable cell counts on each pooled sample. Centrifuge the pooled samples at 250 x g for five minutes.

Then aspirate the supernatant and resuspend the cells in one to three milliliters of room temperature as stem cell maintenance medium supplemented with 10 micromolar ROCK inhibitor Y27632. After calculating the required number of cells needed to achieve the desired seeding density, resuspend the cells to the appropriate concentration and dispense them into the pre-coated plates. The total volume per well should be one milliliter for a 24-well plate and 0.1 milliliter for 96-well plate.

Gently rock the plates from side to side and back and forth to ensure an even cell dispersion and place the seeded plates into the incubator, rocking them back and forth and from side to side. Prepare media for definitive endoderm induction and for the subsequent hepatic progenitor cell specification differentiation as described in the text manuscript. On the day one of the differentiation, remove the spent medium from the wells and replace it with stage one medium one.

On days two, three, and four, remove the spent medium and feed each well with stage one medium two. On day five, fix the wells intended for definitive endoderm differentiation analysis. For the remaining wells, remove the spent medium and feed each well with stem diff hepatic progenitor medium.

On day 10, harvest cells for hepatic progenitor differentiation analysis or proceed with further hepatocyte like cell differentiation. To characterize the hepatic progenitor differentiation cultures, use immunostaining to detect expression of definitive endoderm specific markers on day five and hepatic progenitor specific markers on day 10 and quantify the percentage of HNF4 alpha positive cells. On day 10, also measure alpha fetoprotein and albumin secretion via ELISA.

This protocol was used to differentiate hepatic progenitor cells from both human embryonic stem cells and human induced pluripotent stem cells. At day five of the differentiation protocol definitive endoderm specification was assessed via Sox 17 expression. In both cell lines, Sox 17 was highly expressed with 80 and 87.8%of Sox 17 positive cells for H9 and P106, respectively.

At day 10, hepatic progenitors displayed a cobblestone-like morphology. In addition, hepatic progenitor specification was assessed for HNF4 alpha, AFP, albumin, and cytokeratin 19 expression. Both hepatic progenitor cultures expressed fetal hepatic markers, such as HNF4 alpha, AFP, and CK 19.

Alpha fetoprotein secretion was detected at day 10 in both cell lines while albumin secretion was not detected with ELISA. Cell number variability and hepatic progenitor differentiation efficiency was assessed by quantifying HNF4 alpha expression. At day 10, hepatic progenitors showed no significant variability across rows with over 95 and 97%of HNF4 alpha positive cells per well for H9 and P106, respectively.

An even cell distribution prior to the start of differentiation is key to ensuring a homogenous population of hepatic progenitor cells. To achieve this, gently rock the plates side to side and back and forth to ensure even cell dispersion. Hepatic progenitor cells produced by this protocol can be further differentiated to hepatocyte-like cells for other assays, offering a reproducible and standardized tool for disease modeling or drug screening.