新規の表面マーカーの組み合わせで識別離および肝前駆サブセットの濃縮

The overall goals of this liver digestion protocol are to assess the various liver progenitor subsets by flow cytometry and to isolate these cells to a high purity using a novel surface marker cocktail for further downstream analysis. This method can help answer various questions about the biology of liver progenitor cells, for example, what are their subset specific features and what are their distant cause during liver injury. The main advantage of this technique is that it allows the isolation of progenitor cells for high purity and viability which is important for the in-vitro analysis.

Although this digestion technique provides insight into progenitor cell biology, it can actually be utilized for the isolation of hematopoietic and endothelial cell populations. To prepare a single cell preparation from a mouse liver, transfer the lobes onto a dried petri dish and use a scalpel to cut the tissues into approximately two millimeters cubed homogenous cubes. Transfer the pieces into two 15 milliliter conical centrifuge tubes per liver containing 2.5 milliliters of digestion buffer each.

And place the samples in a 37 degrees Celsius water bath with gentle shaking at five and 10 minutes. After 15 minutes of digestion, use a 1, 000 microliter pipette with a cut tip to gently titrate the liver pieces. Then return the tubes to the bath and allow the pieces to settle for two minutes.

Next, filter two approximately 700 microliter aliquots of the supernatant through a 100 micron strainer pre-wet with 800 microliters of collection buffer. And replace the removed supernatant with fresh digestion buffer. When the liver tissue is no longer visible, pass all of the supernatant from the digestion tubes through the filter.

And collect all of the cells by centrifugation. Resuspend the pellets in one milliliter of ammonium chloride potassium lysis buffer at room temperature stopping the lysis with five milliliters of fresh collection buffer after one minute. Collect the cells with another centrifugation.

Then, carefully aspirate the supernatant without disturbing the loose pellet. And resuspend the cells in four milliliters of fresh collection buffer on ice. To determine the liver sample cell numbers by flow cytometry, first, mix 20 microliters of the liver cell suspension with 174 microliters of PBS in a 1.5 milliliter polystyrene tube and six microliters of propidium iodine.

Add counting beads that allow for cell quantification and load the bead and cell mixture onto the flow cytometer. Gate on the side scatter by forward-scatter parameters to avoid debris. Excluding the doublets via a forward-scatter height versus forward scatter area gate and the propidium iodine positive dead cells.

Then, load a 30 microliter sample onto the flow cytometer and record the events. To stain the liver cells for flow cytometric analysis of the progenitor subsets, transfer 2x10^5 aliquots of the cells into 1.5 milliliter reaction tubes. And pellet the cells by centrifugation.

Resuspend the pellets in Fc block for five minute incubation on ice. Then add 50 microliters of the cell-surface staining antibody cocktail of interest to the appropriate tubes for a 20 minute incubation on ice protected from light. At the end of the incubation, wash the samples in 400 microliters of staining buffer per sample.

And resuspend the pellets in 100 microliters of the appropriate secondary antibody cocktail for 20 minutes on ice protected from light. Then wash the cells with another 400 microliters of staining buffer. And resuspend the pellets in 300 microliters of staining buffer containing propidium iodide.

Measure the sample using a flow cytometer. To enrich for the progenitor cell population by magnetic microbead-based separation, transfer 1.5-2x10^6 cells into the appropriate number of 15 milliliter conical tubes and collect the cells by centrifugation. Resuspend the pellets in 400 microliters of HBSS supplemented with BSA.

And add 40 microliters of anti-CD31 Microbeads and 30 microliters of anti-CD45 Microbeads for 15 minute incubation at four degrees Celsius. Next, wash the cells in five milliliters of HBSS plus BSA. And resuspend the pellets in 100 microliters of dead cell removal beads for a 15 minute incubation at room temperature.

While the cells are incubating, equilibrate one appropriately-sized positive selection column per liver with three milliliters of HBSS plus BSA. Then add 900 microliters of HBSS plus BSA to the cells and filter the cells through a 100 micron polyamide filter mesh. Load the filtered bead-incubated cells onto the columns collecting the eluate in the centrifuge tube.

When all of the cells have flowed through the column, wash the columns three times with three milliliters of HBSS plus BSA. Pellet the cells by centrifugation and resuspend the cell pellet in 100 microliters of rinsing buffer and Fc block. Pull the cell pellets from four to six livers or up to less than 1x10^6 negatively-selected cells.

After five minutes, label the cells with anti-GP38 biotinylated antibody for 10 minutes on ice. At the end of the incubation, collect the cells by centrifugation for resuspension in 400 microliters of rinsing buffer and five microliters of Anti-Biotin Microbeads. After 15 minutes at four degrees Celsius, immediately wash the cells in five milliliters of rinsing buffer.

And resuspend the pellet in one milliliter of fresh rinsing buffer. Place the resuspended cells on a chilled 15-rack and start the automatic magnetic separation. Then centrifuge the flow-through and resuspend the pellets in the appropriate solution for the planned downstream experimental analysis.

Gating on the cells that are negative for CD45, CD31, and asialoglycoprotein one receptor selects for the liver progenitor cells which can then be grouped according to their glycoprotein 38 and CD133 expression. The gp38 positive CD133 positive and the gp38 negative CD133 positive cells represent the most abundant cell populations among the CD45 negative CD31 negative ASGPR1 negative cells in the healthy adult mouse liver with apparent differences in their expression of various surface markers previously associated with progenitor cells. Magnetic-microbead-based isolation of these progenitor cells, as just demonstrated, results in an over 90%purity of the liver cells with a high degree of viability.

When attempting to isolate these uncommon cells, it is important to take into consideration the enzymes used during the liver digestion steps as different enzymes can preferentially yield specific progenitor subpopulations and greatly reduce CD133 expression levels. It's important to remember that the cell purity and yield depend on the gentle preparation of the liver single cell suspensions. And that the high amount of cellular debris and dying cell contamination can negatively effect the success of this protocol.