Derivazione e caratterizzazione di un free-Transgene umana indotta pluripotenti staminali Cell Line e Conversione in definite condizioni clinico-grade

The overall goal of the following experiment is to derive lentiviral based reprogrammed factor free human-induced pluripotent stem cells and convert these cells into putative clinical grade conditions. This is accomplished by first reprogramming adult dermal human fibroblasts with a single ex sizeable lentiviral stem cell cassette or stem car into HI PSCs. Next, the reprogrammed HI PSCs are exposed to adeno Cree purr virus for full excision and removal of stem car from the host genome.

Then genomic DNA extraction from the feeder free HI PSCs, followed by agel analysis confirms proper stem car excision. Finally, the factor three H IPCs are converted into putative GMP grade conditions by a slow conversion methodology into fully defined xeno free conditions. Ultimately, viral based reprogramming of human adult dermal fibroblasts and full conversion of the H IPCs into clinical grade conditions underscores the broad applicability of this methodology for reprogramming adult human cells and converting them into clinically relevant conditions.

The implications of this technique extend toward clinical applications of human induced pluripotent stem cells as they’re completely factor free and converted to clinical grade conditions, making them compatible with human therapeutics. Visual demonstration of this method is critical as reprogramming excision and clinical grade conversion steps are difficult to learn because of the main intricate and specific procedures that must be followed to yield factor three and clinical grade human induced pluripotent stem cells To begin thaw. Adult dermal human fibroblasts or HAFs in a 37 degree Celsius water bath for two minutes.

Taking care not to submerge the top of the vial in the water. Place the one milliliter slurry of cells into a 15 milliliter chronicle vial to resuspend the cells and dilute out the DMSO in a dropwise fashion. Slowly add four milliliters of pre warmed huff medium centrifuge the violet 200 times G for 10 minutes of room temperature.

Then aspirate the sup, nacent and reus. Suspend the cells in medium to 100, 000 cells per milliliter into one well of a six well plate preco to 0.2%gelatin. Add two milliliters of the cell suspension, then add two milliliters of suspension to a second.

Well, to be used for measuring multiplicity of infection or MOI rock the plate side to side and back and forth to evenly distribute the cells before incubating them overnight at 37 degrees Celsius and 5%carbon dioxide in a humidified chamber. 24 hours later after counting the cells thaw out two times center the eight transduction units or S of equivalent lentiviral concentrate and use huff medium to dilute it to one times 10 to the eight ts per milliliter using half medium supplemented with eight micrograms per milliliter of transfection reagent. Transduce the cells at a 10 multiplicity of infection ratio.

Mix the transduce wells evenly and incubate overnight approximately 24 hours after lentiviral transduction. Remove the huff medium and replace it with standard human pluripotent stem cell or PSC medium. Change the medium daily on days two through six on the six day into two 0.2%Gelatin coated 10 centimeter dishes plate 1.5 to 1.75 times 10 to the six mouse embryonic fibroblasts derived from complement component factor one or CF one mice in half medium on day seven.

Add room temperature trips into one well of a six well plate of the reprogrammed fibroblasts. She gently lift off the cells and spin a 100 times G with fresh human PSC medium plate all the cells at a one to 16 ratio on two 10 centimeter CFI meth plates. Distribute the aggregates in a clockwise spiral movement and incubate for approximately 48 hours before replacing the medium.

After three to four weeks, use a 21 gauge needle to pick eight to 10 individual pieces of specific colonies with typical ESC like morphology and subclone them into individual wells of CFI MFS after expansion. Confirm that the cells are pluripotent by embryo body formation and marker expression analysis after verifying the stem car integration site as described in the accompanying text protocol. Excise stem car by first aspirating off the old human PSC medium into three milliliters of standard PSC medium combined.

45 microliters of concentrated adeno Cree pur or virus with eight micrograms per milliliter of transfection agents. Then add to the cells and incubate for 24 hours after the incubation aspirate the viral supinate induce human PSC medium to wash the cells twice. Then add HPSC medium with two micrograms per milliliter of pure mycin and replace daily after five days, subclone the remaining colonies and expand them as described earlier in the video.

Next thaw one vial of pre Eloqua basement membrane matrix on ice and diluting cold basal medium to a final concentration of one to 30. Use one milliliter per well to coat the desired number of wells of a six well plate and let the plate sit at room temperature for one hour. Then using an 18 gauge needle, crosshatch at least 20 to 30 colonies from a well previously coated with mes.

Take care to reduce uplifting mes and transferring the cells from the plate. After the one hour incubation, aspirate the matrix from the coated well with a 200 microliter pipette. Remove pieces of the colonies by gently scraping them from the old plate and carefully place them into three milliliters of freshly combined medium, one in a matrix coated plate.

After incubating for 48 hours, change the medium daily when the post excised and feeder free converted HI PSCs reach 80%co fluency. Use a commercial DNA extraction kit to extract the genomic DNA using the primers shown here that are specific for exogenous integrations of the stem car lentivirus. Analyze the DNA by PCR for proper excision to transition the post excised HI PSCs to GMP grade conditions.

Mix HPSC combined medium one and clinical grade GMP compliant medium or combined medium two at a ratio of 80 to 20 prewarm to 37 degrees Celsius. Aspirate the old combined medium one from the cells and replace with three milliliters of the new warm medium solution. Replace the medium daily for three days passaging according to the text protocol to maintain proper colony size and density on day four, replace the medium with a 50 50 mixture of warm combined medias one and two incubate for three days.

Replacing the medium daily on day seven, switch to a 20 to 80 ratio of warmed combined medias one and two respectively and replace daily for three days. On day 10, switch to warm, 100%combined medium two and change daily. The cells are now in completely defined xeno free medium.

Next, after using a defined xeno free substrate to coat one well of a six well plate replace the xeno free medium in a well of confluence cells with fresh medium containing one x rock. After incubating for one hour, use an 18 gauge needle to mechanically the cells into the freshly prepared. Well allow the cells to incubate undisturbed for two days to allow for maximum attachment before characterizing the cells according to the text protocol.

This figure shows a representative picture of three different pre excised H-I-P-S-C lines after reprogramming with the stem car approach on a layer of mes shown here is the post excision reverse transcription PCR gel showing one particular sub clone completely free from the stem car lentivirus as evidenced by the lack of an amplicon band specific for a particular sequence endogenous to stem car. In this figure, pre-con converted IPCs on a xeno containing matrix and post excise xeno free GMP grade H IPCs on synthetic matrix are shown quantitation of standard pluripotency associated factors, including SOX two, OCT four, and nanog. Using QPCR is illustrated here.

Finally, as seen here, pre-con converted and post converted HI PSCs to GMP grade conditions were tested through flow cytometry for the sialic acid and glycol neuronic acid indicative of non-human antigens. After watching this video, you should have a good understanding of how to reprogram human adult dermal fibroblasts with the stemco reprogramming virus, excise the virus out of the genome, and then fully convert these HI PSCs into clinical grade conditions fully compatible with human therapeutics. Don’t forget that working with staca, a third generation antiviral vector can be extremely hazardous and precautions.

Such a proper PPE and a VSL two plus tissue culture room should be used while performing this procedure.