The overall goal of this procedure is to generate human natural killer or NK cells from blurry, potent stem cells. This is accomplished by first generating hematopoietic progenitor cells. In the second step, the hematopoietic progenitor cells are differentiated into NK cells through the use of defined cytokines.
Next, NK cells can be tested in a variety of functional assays. In the final step, dual imaging is used to monitor the ability of NK cells to kill tumor targets in vivo. The main advantage of this technique over traditional existing methods such as stromal cell mediated differentiation or regular EB formation, is that the spiny B system provides a completely defined feeder free approach for the large scale expansion of progenitor cells that can be differentiated into natural killer cells.
The implications of this technique expand towards the treatment of chronic viral infections, as well as cancer, as NK cells have been shown to be important effector cells in both of these diseases. To prepare for embryo body or EB plating, first pipette 150 microliters of sterile water into the 36 outer wells of a round bottom low attachment 96 well plate to minimize evaporation. Next, aspirate the medium off of the embryonic stem and induced pluripotent stem cell cultures and add one milliliter of Prewarm trip.
Select place the trip alley containing plates into a 37 degrees Celsius incubator, and then after five minutes, gently pipette the cells off of the plate, transfer the dissociated cells to a conical tube and pipette the cell solution up and down to break apart any clumps. Now dilute the trip Ali with one equal volume of BPEL media and at least one equal volume of DPBS and spin down the cells for five minutes at 480 times G and eight degrees Celsius. Then remove the supernatant and resuspend the pellet in five milliliters, BPEL media plus five milliliters DPBS.
After spinning down the cells again, resuspend the pellet in five to 10 milliliters of BPEL media and then pass the cells through a 70 micron filter into a fresh 50 milliliter conical tube to remove any clumps. After counting and spinning down the filtered cells Resus, suspend the palate at three times 10 to the fourth cells per milliliter plate, 100 microliters of cells into each of the inner 60 wells of the previously prepared 96 well plates, and then spin the 96 well plates for four minutes at 480 times G and eight degrees Celsius. Then carefully place the plates in a 37 degrees Celsius incubator using a multi-channel pipette sent to 100 microliters.
Begin this next step by transferring six spin EBS to each well of a 24 well plate. Then add 400 microliters of NK differentiation media containing cytokines to each well to bring the total volume in each well to approximately one milliliter. Incubate the cells for 28 days, feeding the cultures every five to seven days with NK differentiation, media containing cytokines.
At day 28, the NK cells will express a mature phenotype following a transfer to NK cell differentiation culture. To test NK cell cytotoxicity, perform a four hour chromium release assay to generate large numbers of NK cells for the in vivo experiments. Co-culture the cells with artificial antigen presenting cells using the protocol previously demonstrated by Sochi et al.
Begin this step by Resus suspending the desired number of tumor cells in 200 microliters of is cov modified delco medium, or IM DM supplemented with 20%FBS. Then using a 27 or 28 gauge needle, inject the tumor cells subcutaneously into the upper left thorax of each six to eight week old, non-obese diabetic. Severe combined immunodeficiency with gamma chain knockout mouse.
Allow the tumor cells to engraft for four days. The cells should form a bubble under the skin of the mouse. Then resuspend the desired number of HESC derived NK cells in 300 microliters of IMDM, supplemented with 20%FBS without antibiotics.
Next, using a new 27 or 28 gauge needle intraperitoneal, inject the HESC derived NK cells into each mouse with a tumor. Then to monitor the engraftment of the cells by bioluminescent imaging, inject each mouse peritoneal with 120 microliters of D luciferian. After anesthetizing the mice, place the animals in an IVUS spectrum within 10 minutes after injection of the D luciferian, secure each mouse on its back using Velcro, and then set the IVUS machine to the correct imaging platform.
Now set the benning to medium and the F stop to one, and then acquire the image for one minute to perform fluorescent imaging for turbo FP six 50 positive cells. First, use the imaging wizard to set up an emission scan to measure the probe of interest, as well as background signal from the list of probes. Choose input EM ex and enter 605 for the excitation and 660 to 720 for the emission for the tumor signal.
Then select add probe for the background signal. Set the excitation to 570 and the emission to 640 to 720. Filters can also be set through the filter config button.
Finally, allow the mice to fully recover from the anesthesia before transporting them back to the animal facility. The images can be analyzed using the living image software package version 4.2. Day 11, spin EBS contain high percentages of progenitor cells expressing CD 34, CD 43, CD 45, and CD 31.
High levels of CD 34 and CD 45 allow direct transfer to NK cell conditions without the need for sorting or for supporting stromal cells. NK cells can be detected by flow cytometry. After four weeks of culture.
Cells within the lymphocyte gait are analyzed for the expression of CD 56. CD 56 positive cells can then be further analyzed for co-expression of markers such as CD one 17, CD 94, or NK P 46. NK cells maintain the expression of reporter genes that were modified into the parent E-S-I-P-S line and therefore can be followed in vivo.
Here, the IVUS spectrum has been used to demonstrate the simultaneous imaging of both NK cells and tumor cells. In the same mouse bioluminescent imaging of Firefly Luciferase, HESC derived nnk cells on day zero seven and 14 after NK cell injection is shown fluorescent imaging of turbo FP six 50 positive K 5 6 2 cells on day zero seven and 14. After NK cell injection is shown here, a sequence showing the tissue autofluorescence and fluorescence signal from the turbo FP six 50 positive cells generated.
Using the spectral unmixing feature in living image software is shown. These mice are non injection controls and show only the background signal. While attempting this technique, it is important to maintain high quality ES and IPSL culture, as well as performing DYS spinny b differentiation as described.
