This video demonstrates a procedure for isolating liver stem cells to study clonal expansion using CD 1 33 cell surface expression following chronic liver injury. First, the liver's removed from a mouse with chronic liver injury and digested to produce a single cell suspension. Red blood cells lysis buffer is then added to remove the red blood cells and the solution is applied to a magnetic CD 45 antibody containing column to remove CD 45 leukocytes.
The CD 45 depleted cells are then sorted to enrich CD 1 33 positive liver stem cells. Finally isolated CD 1 33 positive liver stem cells are cultured and expanded. In vitro gene and protein expression analysis demonstrate that the isolated CD 1 33 positive stem cells are able to differentiate into both hepatocytes and osteocytes.
The main advantage of this technique over existing methods such as colocalization of markers using immunohistochemistry techniques is that single cells can be isolated for clonal expansion and functional analysis To confirm stem and pro generator cell phenotypes, demonstrating the technique will be myself and he dang. A graduate student in my lab. Prepare all buffers in media 24 hours in advance and store them at four degrees Celsius until use all solutions.
Media, instruments, filters, and tubes should be sterile and handled with sterile technique. To reduce the risk of contamination, wipe the abdominal area of a euthanized mouse with 70%ethanol. Using sterile instruments, open the abdominal cavity and explain the liver on block.
Remove the gallbladder from the explanted liver, then transfer the whole liver to a laminar flow hood in a closed sterile dish in a laminar flow hood, use a sterile razor blade to mince the liver with a combination of multiple horizontal and vertical cuts. For one minute, divide the minced liver into four parts and place each part in its own 15 milliliter tube with 10 milliliters of enzyme digestion solution. Then place the tubes in a 37 degree Celsius water bath for 45 minutes in a shaker set to one to two cycles per second.
Following the incubation wipe tubes with 70%ethanol. Then under a laminar flow hood, pipette the digested liver pulp through 70 micron mesh Filter and collect the flow through in a sterile dish to remove the parenchymal fraction which contains the larger hepatocytes. Rinse the filter with two milliliter aliquots of sterile supplemented D-M-E-M-F 12 medium.
Use the rubber end of a syringe plunger to mash the digested pulp through the filter. Repeat this process five times to make total volume of approximately 20 milliliters. Divide the filtrate into two equal 15 milliliter tubes, then centrifuge at 50 times G for one minute at four degrees Celsius.
Following the spin. Transfer the SUP natin to a tube labeled snat number one and discard the parenchymal palt. Next to collect the non parenchymal fraction, which contains the stem and progenitor cells centrifuge SUP natin number one, add 50 times G for one minute.
Transfer the supernatant to a tube labeled supernatant. Number two, and again discard the pellet. Then centrifuge supernat number two at 50 times G for one minute.
Following the spin, transfer the supernatant to a tube labeled supernatant number three, and discard the pellet. Finally, centrifuge final SNAT number three for 180 times G for eight minutes to obtain non parenchymal fraction following the spin, discard the supernatant and proceed immediately to the next step of the protocol in which the red cells and the pellet are laced. The red cell lysis, buffer and mill tene buffer used for this part of the procedure should have been prepared the night before and stored at four degrees Celsius according to the instructions in the accompanying written document.
Working in a laminar flow hood with the cells and solutions on ice. Resus suspend the non parenchymal pellet from step above in one milliliter of one x diluted red blood cell lysis buffer and transfer it to a five milliliter tube. Cap the tube and gently vortex it for five seconds.
Incubate for 15 minutes of four degrees Celsius. Protected from light after 15 minutes have passed. Centrifuge the tube at 200 times G for five minutes following the spin to scar the supernatant which contains lysed red blood cells and resus.
Suspend the pellet in one milliliter ice, cold and sterile. Melt e buffer centrifuge of 200 times G for five minutes. After the spin, discard the supernatant and resuspend the pellet in one milliliter ice cold and sterile.
Milton e buffer. Then remove 10 microliters of this cell suspension and add 10 microliters stripe am blue. Count the non parenchymal cells using a hemo cytometer to perform the CD 45 hematopoietic cell depletion from the non parenchymal fraction.
Begin in the laminar flow hood by adjusting the concentration of the cells to 10 to the seventh to 10 to the eighth. Total cells in 100 microliters of melty buffer. Be sure to keep the cells and buffers cold throughout the entire procedure.
Next, apply 20 microliters of mil, any CD 45 microbead antibody per 10 million cells and incubate at four degrees Celsius for 15 minutes. Following the incubation, add an additional two milliliters mil, any buffer and centrifuge 200 times G for five minutes. After the spin, remove the snat and resuspend up to 10 to the eighth cells in one milliliter.
Milt, any buffer in a laminar flow hood, place a Milt, any LD magnetic column in a magnetic holder. Place a sterile five milliliter tube below the filter to catch the filtrate. Then load two milliliters.
Melt any buffer to perform a filter pre-wash. Once the wash is complete, load the cells onto the column. Once the cell suspension is within the column, add one milliliter melt e buffer to wash the column, perform this wash two more times.
Do not use the plunger provided with a column to increase the speed of filtration. Once all of the washes have been performed, centrifuge the collected filtrate of CD 45 depleted non parenchymal cells at 200 times G for five minutes. Discard the column with a retained cd 45 positive cells.
Next flow cytometry is performed to isolate CD 1 33 positive cells, resuspend cells in Milt, any buffer, 10 to the seven cells per 100 microliters aliquot 100 microliters into three tubes to the first tube. Add two microliters of CD 1 33 PE conjugated antibody to the second tube. Add IgG PE conjugated antibody as a control.
The third tube of cells will serve as the unstained control incubated four degrees Celsius for 15 minutes in the dark following the incubation resuspend in two milliliters of staining buffer centrifuge 200 times G for five minutes. Discard supernatant and resuspend the pellet in one milliliter Milton e buffer. Next on the flow cytometer use unstained cells and IGPE stain cells to adjust the sorting parameters for optimized gating of CD 1 33 positive cell population.
Our FICO ERYN or RPE can generally be used with any flow cytometer that has a laser that emits 488 nanometers. Finally, isolate the CD 1 33 positive cell population using CD 1 33 positive gait and collect the cells in sterile filtered oval cell medium. The cells can now be cultured and clonal Isolation can be performed followed by real-time PCR to assess by potential status CD 1 33.
Positive liver stem cells were isolated from wild type and MAT one A knockout mice as described in this video as seen here. CD 1 33 positive cells make up 0.4%of the highly enriched CD 45 depleted non parenchymal control fraction derived from uninjured liver from wild type mice in the genetic knockout MAT one A minus minus, which is a model of chronic liver injury. The CD 1 33 positive population expands tenfold or more in the same highly enriched fraction.
Following sorting cells were cultured and clones were expanded.Shown. Here are phase contrast images of four colonies derived from single CD 1 33 positive cells expanded on laminin coated 96 well plates. These cells are seven days post isolation and demonstrate small compact epithelial cell colonies at day seven when colonies are approximately 25 cells.
RNA was isolated from the cells and gene expression of the cholangio site marker CK 19 was analyzed as can be seen in these R-T-P-C-R experiments. Gene expression demonstrates expression of hepatocyte marker albumin and cholangio site marker CK 19 confirming by potential status of CD 1 33 positive cells several weeks after CD 1 33 positive initial single cell isolation and expansion in vitro one times 10 to the six cells from the mat one, A minus minus model are injected subcutaneously into immune deficient nude mice. Arrows indicate tumors growing four weeks after the cells are injected.
CD 1 33 positive cells from toxin induced chronic liver injury such as CCL four or 0.1%DDC diet do not form tumors. Tumor formation of vivo is used to identify malignant potential or cancer stem cell phenotype within the stem cell population. Once mastered, this technique can be completed in eight hours if properly performed.
Following this procedure, adding additional facts markers such as EPAM or other facts. Techniques such as the side population flx can be performed in order to answer additional questions like refining the surface markers of liver progenitor cell populations. After watching this video, you should have a good understanding of how to isolate liver stem and progenitor cells using flow cytometry.
Don't forget that working with animal tissue can be hazardous, and precautions such as wearing gloves and proper laboratory techniques should always be taken while performing this procedure.
