The overall goal of this procedure is to explain how to derive endothelial colony forming cells or e CFCs from human umbilical cord blood. This is accomplished by first plating mononuclear cells from the umbilical cord blood, and then cloning and expanding colony outgrowths of E CFCs. The second step is to characterize the cells in vitro using a novel single cell assay technique for clonal proliferative potential.
Finally, the cells are characterized in an in vivo vessel forming assay through the production and implantation of gels containing E CFCs in nod skid mice. Ultimately, the in vivo formation of de novo host blood filled vessels can be evaluated by h and e staining of the ECFC containing cellular implants. The main advantage of these techniques over existing methods is that these methods allow for the examination of the proliferative potential of endothelial cells at a single cell level.
The implications of this technique could be extended towards finding diagnostic and therapeutic tools for diseases such as feral arterial disease or PAD because this technique allows us to examine the proliferative potential and genic capacity of a patient's endothelial cells. Before beginning the procedure, add one milliliter of rat tail collagen, type one into each well of a six well tissue culture plate, and then incubate the plate overnight on the day of the procedure, aliquot the cord blood in 15 milliliter units into 50 milliliter conical centrifuge tubes. Then add 20 milliliters of PBS to each tube and pipette the mixture up and down several times.
Next, draw 15 milliliters of fial PA into a pipette place the tip of the pipette at the bottom of the tube of diluted blood and carefully underlay the alpac centrifuge the tubes for 30 minutes at 740 times G at room temperature without the deceleration brake setting. After the cell separation, use a transfer pipette to remove the hazy layer of low density mononuclear cells located at the interface between the fial peak and the diluted plasma. Dispense the collected cells into a 50 milliliter conical tube containing 10 milliliters of E GM two medium and pellet the mononuclear cells for 10 minutes at 515 times G at room temperature with the high deceleration brake setting, carefully aspirate and discard the supernatant.
Then after washing the pelleted cells with EGM two twice resus, suspend them mononuclear cells in EGM two at 1.25 times 10 to the seventh cells per milliliter. Now pipette four milliliters of the mononuclear cell suspension into each well of the collagen coated plate and incubate the cells until colonies form on the first day. After incubating, slowly remove the spent medium from the wells with a pipette without disturbing the loosely adherent cells.
Then carefully add four milliliters of E GM two to the wells and return the plate to the incubator. Colonies typically appear between days five and 14 the day before the procedure. At 50 microliters of rat tail collagen.
Type one to each well of a 96 well plate as before and incubate the plate overnight on the day of the procedure. Begin by washing cells that have been passaged only two to three times twice with PBS. After aspirating the final wash of PBS place a sterile gel coated cloning cylinder around each colony, pressing each cylinder firmly against the plate using sterile forceps.
Add two to three drops of warm triple E express into each cloning cylinder and incubate the cylinders for three to five minutes until cells begin to detach. After the cells have begun detaching at approximately 250 microliters of EGM two medium into the center of the cylinder and pipette up and down to generate single cell suspension. Now transfer the single cell suspension from each cloning cylinder separately into an individual micro centrifuge tube.
Infect the cells in a six well plate with EGFP expressing lentiviruses and collect the cells expressing EGFP by fluorescent cytometry. Then incubate the EEG FP positive cells and EGM two in the collagen coated plates. When the E CFCs are ready, collect them Using trip E express as just demonstrated and resus suspend the cells and e GM two medium to a final concentration of approximately 10 to the sixth cells per milliliter.
Then using a flow sorter with a low flow rate of 20 cells per second sort one single EGP positive ECFC per well into the previously prepared collagen coated 96 well plate adjust the final volume of each well with EGM two to approximately 200 microliters per well. Then use an inverted fluorescence microscope to ensure that each well contains only one cell. Incubate the plate over a period of two weeks with two media changes on day 14 of culture.
Wash each well with 100 microliters of PBS before fixing the cells with 100 microliters of 4%paraform aldehyde for 30 minutes. For e CFCs that are not expressing EGFP add cyt reagent, a green fluorescent nuclear dye following para formaldehyde fixation and incubate at four degrees Celsius overnight. Use a fluorescence microscope to visibly quantify the number of expanded endothelial cells as demonstrated here wells with an endothelial cell number of two to 50 are considered endothelial cell clusters.
Wells with 51 to 2000 cells are low proliferative potential e CFCs and wells with 2001 or more are considered high proliferative potential e CFCs. After detaching the cells with triple E express as before, obtain a viable cell count of the cell culture by hemo, cytometer and trian blue. Transfer 2.4 million viable cells into a 50 milliliter conical tube, and then pellet the cells by centrifugation while the cells are being spun down.
Mix the components for the gel matrix thoroughly and then adjust the pH to 7.4 with sodium hydroxide while keeping the solution on ice. After discarding the supernatant resuspend the pellet in 360 microliters of warm e GM two. Then add 840 microliters of the gel matrix solution to the cells and mix the solution carefully until the cells are thoroughly suspended in the gel Transfer this one milliliter cellularize gel suspension to one well of a 12 well tissue culture plate and incubate the plate until the gel polymerizes.
Then before incubating overnight, gently cover the gel with two milliliters of warm EGM two the next day and immediately before implantation. Use iris scissors to dissect the gel into two equal pieces and return the gel pieces to the same culture well containing e GM two medium. After confirming sedation by toe pinch shave the lower abdomen of a five to six week old anesthetized nod, skid mouse and thoroughly clean the surgical site by rotating alcohol and betadine pads.
Next, use sterile sharp iris scissors to make an approximately five millimeter incision in the lower quadrant of the abdomen, exposing the subcutaneous space between the skin and the abdominal muscle.Blunt. Dissect the dermal layer from the abdominal muscle in two different areas of the abdomen to create 15 to 20 millimeter wide pockets, one leading superior to the upper abdominal quadrant and one into another area of the abdomen. Then lifting the dermal layer just coddle to the incision.
Insert one half piece of the gel into one abdominal pocket in the other half piece of gel into the other abdominal pocket. After the gels have been inserted, close each incision with two to three stitches using five oh polypropylene sutures on a cutting needle. Label the cage cards and perform post-surgical monitoring and analgesia as per the institutional and protocol requirements.
After sacrificing the animal on day 14 of implantation, swab the mouse's abdomen with alcohol pads and cut the skin coddled to the original incision line. Then excise flaps of skin coddled to the probable location of the gels. Cuts circumferentially around the gels to remove each implant, and then place excised implants into the zinc fixative.
Using this ECFC derivation technique, we have observed outgrowth of primary colony formation as early as day five. Note the cobblestone morphology of these day five ECFC cultures as indicated by the arrowheads. Expanded colonies express endothelial antigens, but do not express hematopoietic antigens.
As shown in this representative in vitro phenotypic assessment of endothelial and hematopoietic cells, immunophenotyping has revealed that e CFCs express endothelial antigens such as CD 31, CD 34, CD 1 44, CD 1 46, FL one, flick one, flip four, and NRP two, but did not express hematopoietic antigens, CD 45 CD 14, CD 11 BC Kit CXCR four or CD 1 33. Importantly, as represented in this bar graph expanded colonies display a complete hierarchy of clonal proliferative potential at the single cell level that range from clusters of two to 50 cells up to colonies of greater than 2001. These micrographs of colonies stained with cyt, a green fluorescent nuclear dye were obtained after cord blood derived E CFCs were cultured at the single cell level for 14 days.
Moreover, e CFCs form humanized blood vessels that are perfused with host red blood cells when implanted into immunodeficient mice. In this h and e staining of cord blood derived ECFC containing CELLULARIZE gel implants, microvessels filled with host red blood cells that formed in the collagen fibronectin gel after 14 days of implantation can be observed. Here, the anti-human CD 31 staining in brown further confirms the human origin of these vessels.
After watching this video, you should have a good understanding of how to derive e CFCs from human umbilical cord blood, and of how to clone, expand, and characterize the functionality of ECFC colonies After its development. This technique paved the way for researchers in vascular endothelium to explore the endothelial clonal proliferative potential and genic capacity of endothelial cells that are obtained from multiple sources.
