The overall goals of this procedure are to isolate stem ness transferring extra cellular vesicles and exosomes from stem cells. And to assess the stem ness transferring ability of these nano-particles. The meso can answer key questions in the stem cell field about weather stem cell derive in trans cell or vesicle.
Or exosome can transfer stem cell property to non stem cell. The main advantage of this technique is that the induced stem cell derive in trans cell vesicle. And the exosome can be used to directly reprogramming non stem cell into stem cell.
Demonstrating the procedure will be postal researcher LeMonsier, with surgical assistant, Pei-Ling, Wen-Tin, and Shih-Yin, all from my laboratory. Begin by seeding one point two times ten to the sixth non-adherent/mesenchymal mammary epithelial cells, or NAMECs in 12 milliliters of medium per 15 centimeter dish for a four day culture in a cell culture incubator. On day four, harvest the culture medium for table top centrufigation.
Then transfer the supernatent into a conical tube for a second table top centrifugation. Next, transfer the supernatent into a new ultra-centrifuge and perform ultracentrifugation for 30 minutes. At the end of the spin, transfer the supernatent into a new ultra-centrifuge tube and ultra-centrifuge the supernatent again.
Remove the supernatent and re-suspend the extra-cellular vesicle exosome palate in PBS for a third ultracentrifugation. Then, re-suspend the palate in fresh PBS and ultra-centrifuge the cell particles one final time. Remove the supernatent and re-suspend the extra cellular vesicle exosome palate in 100 microliters of PBS.
Then, measure the protein concentration of the extra cellular vesicle suspension by bicinchoninic acid assay. The concentration should be approximately 20 to 40 micrograms per 100 microliters. To measure the concentration and the size of the extra cellular vesicles and exosomes, dilute the cell particles to two micrograms of protein per 100 milliliters of PBS.
And analyze the particles by nanoparticle tracking analysis. To purify the exosomes by density gradient, after the third ultracentrifugation, resuspend the palate in two milliliters of 40 percent iodixanol in PBS. And overlay the cell suspension with sequential two milliliter volumes of 30 percent, 20 percent, 10 percent and five percent iodixanol.
Separate the cell fraction by density gradient ultracentrifugation using a pipette to harvest each of the ten gradient layers at the end of the centrifugation. Then, analyze the presence of exosome markers in each fraction by SDS page and western blot according to standard protocols. Using antibodies against the appropriate exosome markers of interest.
To treat mammary epithelial cells with the extra cellular vesicles and exosomes first, seed two times tent to the fifth flow sorted epcam hi-CD 49F low luminal mammary epithelial cells per plate onto gelatine coated six well plates. Then, treat each cellular culture with two micrograms per milliliter of the NAMEC derived extra cellular vesicles and exsomes. Replacing the cultured medium with fresh extra cellular vesicles and exosomes every two days for ten days.
At the end of the treatment period, place a three week old female anesthetized 57 BL6 mouse in the supine position, and remove the fur on the mid-abdomen. Disinfect the surgery site with three alternating cycles of 70 percent alcohol and povodone iodine. And make a one point five centimeter vertical incision through the skin, along the ventral thorassic inguinal region.
Alternately expose the right and left fourth mammary fat pads. Locate the lymph node in each fat pad and remove the whole gland perancama below the lymph node. Next, use a natural enzyme with prodialitic and cologenalitic activity to detach the extra cellular vesical and exsome treated luminal mammary epithelial cells.
Neutralizing the enzyme activity, with five percent FBS in PBS after ten minutes. Collect the cells by centrifugation and discard the supernatent. After counting, dilute the cells to one times ten to the second to one times ten to the fourth cells per 15 microliters of PBS concentration.
And use a 100 liter microliter glass syringe equipped with a 27 gauge needle to inject 15 microliters of the mammary epithelial cell suspension into each fat pad. Then, close the skin with wound clips. Eight weeks after the injection, make a vertical incision through the skin from the thorassic region to the ingrinal region and expose both the right and left fourth mammary fat pads.
Remove the fourth mammary glans and spread the fat pads onto individual glass microscope slides. Then, transfer the slides to a chemical hood, and fix the pads with collies fixatives overnight at room temperature. The next morning, wash the samples in 250 milliliters of 70 percent ethanol for 15 minutes.
Followed by 250 milliliters of distilled water for five minutes. After the distilled water wash, stain the fat pads with carmine alum overnight at room temperature. Followed by dehydration in ascending 15 minute ethanol incubations.
After the last dehydration, transfer the samples into zylene in a chemical hood until the fat pads become transparent. Then, mount the slides with mounting medium, and use a digital slide scanner to image the fat pads at 2, 400 dots per inch. The size of the isolated vesicles and the ultracentrifuge palate are typically approximately 100 nanometers, as measured by nano particle tracking analysis.
Further, transmission electron microscopy analysis of ultracentrifuge fractions of NAMEC conditioned medium reveals the presences of abundant membrane vesicles. After density gradient separation as just demonstrated, exosome markers can be detected in the 20 percent iodixonal fraction. Flow side emetric analysis of human luminal mammary epithelial cells traded with CFSE labeled namic derived, extra cellular vesicles and exsomes reveals a ten fold higher CFSE signal in the exosome treated cultures.
Indicating the specific uptake of CFSE labeled extra cellular vesicles and exosomes, by the mammary epithelial cells. Further, while the negative control, PBS treated mammary epithelial cells do not exhibit a CFSE singnal, evidence of namic derived extra cellular vesical and exosome uptake by the exosome treated cells can also be observed by confocal microscopy. Notably, the fat pads of mice injected with flow sorted epCAMhi/CD49Flo luminal mammary epithelial cells treated with NAMEC derived extra cellular vesicles and exosomes for ten days acquired the ability to form mammary glands.
After it's development, this tech paved the way for our researcher in the field of stem cell biology. To explore in the usage of the stem cell derived trans cell and vesicle and exosome in regeneration medicine and direct cell reprogramming.
