Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

The overall goal of this procedure is to generate human induced pluripotent stem cells using hair derived keratinocytes. This is accomplished by first isolating hairs from a donor. The second step is to plate down the hairs in order to facilitate keratinocyte outgrowth.

Next, the keratinocytes are maintained and expanded in vitro. The final step is to reprogram the keratinocytes into human-induced pluripotent stem cells using an episomal reprogramming method. Ultimately, human IPS cells can be isolated and characterized using immunohistochemistry to show the expression of pluripotent markers.

The overall advantage of this method over existing methods such as retroviral or lentiviral method, is that by using episomal factor, we are avoiding undesirable transgene. The integrate into the genome in the stem cells that are generated Begin by thawing a vial of extracellular matrix solution on ice Overnight. The next morning, use pre chilled pipette tips to add 200 microliters of the solution to 12 milliliters of chilled DM F 12 medium.

Add one milliliter of the diluted extracellular matrix solution to each well of a 12. Well plate and incubate the plate overnight at 37 degree Celsius to coat the wells with matrix. Next, collect primary human hair from the donor's head by placing fingers close to the root of the hair and plucking the hair in one quick and smooth motion.

Check to confirm that each collected hair contains an intact hair follicle. Place the follicle under a dissecting microscope and determine the growth phase of each hair. Collect five to 10 antigen hairs from each individual to extract keratinous ice.

The antigen phase, unlike the T phase, contains multiple layers of epithelium. Place the hair samples into a Petri dish containing 10 milliliters of an antibiotic mixture for five minutes, and then wash them in PBS for five minutes at room temperature. Next, grasp the hair with sterile forceps and use a pair of sterile scissors to cut off the excess hair shaft, leaving a hair fragment with an intact hair follicle and around 0.5 to one centimeter of hair shaft.

Carefully transfer one hair fragment into each well of the extracellular matrix coated plate. Using the sterile forceps I pet approximately 100 to 200 microliters of serum replacement. Medium dropwise to each well containing hair samples.

Then place the plate in the incubator and allow the follicles to attach overnight at 37 degrees Celsius and 5%carbon dioxide the following day. Gently add another 100 to 200 microliters of serum replacement, medium dropwise to keep the hair samples moist. Check the hair samples daily to confirm that the hair follicles have attached successfully to the plate.

Once attached, gently add an additional 500 microliters of medium to the well. Change media every two days. Preco a six wall plate with collagen.

Type one using a pre-treatment such as a coating matrix kit at 680 microliters of the dilution medium from the kit to each well followed by 6.8 microliters of the coating matrix solution. Shake the plate gently to ensure uniform coating. Allow the coated plate to incubate for 30 minutes at room temperature prior to plating keratinocytes.

Remove the coating solution, dissociate keratinocytes in culture into a single cell suspension by adding 500 microliters of 0.25%tripsin per well and incubate the plates for two to five minutes at 37 degrees Celsius. Once the cells release from the plate, inactivate the trypsin with 500 microliters of serum containing medium and collect the cells in a sterile 15 milliliter tube. Remove the hair fragment using sterilized forceps, pellet the cells and then resus.

Suspend them in fresh keratinocyte growth medium plate 40, 000 keratinocytes into one well of the collagen coated plate in the presence of keratinocyte medium. Change the media every day and passage the cells when the culture is about 80%confluent V code a six wall plate with gelatin by adding two milliliters of a 0.1%gelatin solution into each, well allow gelatin to coat for at least 20 minutes at room temperature. In the meantime, dissociate keratinocytes that are passage six or lower into a single cell suspension by adding one milliliter of 0.25%tryin per well and incubating the cells for two to five minutes.

At 37 degrees Celsius, inactivate the trypsin with one milliliter of media containing serum and collect the cells in a 15 milliliter tube. Pellet the cells and resuspend them in keratinocyte medium. Then plate 100, 000 to 150, 000 keratinocytes per well in a gelatinized six well plate and incubate them overnight on the following day, day zero, check the plate to ensure the cells are 50 to 60%confluent.

Perform a transfection of the episomal reprogramming vectors on the keratinocytes using a ratio of eight microliters of transfection reagent to two micrograms of total plasma DNA as described in the accompanying text protocol. The next day, day one, change the keratinocyte medium and check the transfection efficiency by estimating the percentage of green fluorescent protein positive cells under a fluorescent microscope. A transfection efficiency of 60 to 70%is expected on day two.

Repeat the transfection to boost the transfection efficiency to greater than 90%Then pre-code a 10 centimeter dish with five milliliters of 0.1%gelatin for at least 20 minutes. Plate 4 million mitotically inactivated feeder cells per 10 centimeter dish in FBS medium as described in Conrad Al all and allow the cells to settle and attach overnight in a 37 degree Celsius incubator with 5%carbon dioxide on day three. Harvest the transfected keratinocytes as previously shown, and resuspend them in KSR medium plate.

90%of the keratinocytes from one of the wells into a 10 centimeter dish prepared with feeder cells and cover them with KSR. Medium Isolate human induced pluripotent stem cell colonies by manual dissection from day 21 onwards. Avoid colonies that are closely clustered together and only pick colonies that are clearly separated from others.

For manual dissection, use a 26 gauge needle to cut the colonies into small pieces around 300 to 600 micrometers in length under a dissecting microscope. Transfer the pieces from one colony into a new MEF feeder plate to establish a clonal line of human induced pluripotent stem cells. Once the culture reaches 70 to 80%con fluency with human induced pluripotent stem cell colonies of about 1.5 millimeters in diameter passage, the cells using standard enzymatic packaging methods such as Accutane, sase, or collagenase.

Four, according to the manufacturer's instruction. When following this protocol, recy outgrowths can be observed as early as three days after hair attachment and will continue to proliferate. The keratinocytes shown here were passaged onto a new plate and these cells can be maintained for multiple passages.

This image shows a typical keratinocyte derived human induced pluripotent stem cell colony. After 32 days of reprogramming, some differentiation can occur at the center of the human induced pluripotent stem cell colony. Once manually picked the derived cells typically display high nucleus to cytoplasmic ratio and a defined colony boundary.

They also express a number of pluripotent markers such as OCT four nag and TRA one 60. After watching this video, you should have a good understanding of how to extract keratinocyte from hair and reprogram them into human induced purporting stem cells.