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

The collection of human hair sample from individuals requires ethical approval by the human research ethics committee in the host institutions and should be done in compliance with the institutional guidelines.

1. Isolation of Keratinocytes from Plucked Hair

1. Thaw Extracellular Matrix (ECM) solution (i.e., Matrigel) on ice O/N.
2. Using pre-chilled pipette tips, add 200 µl ECM solution to 12 ml of chilled DMEM/F12 medium. Coat a 12-well plate with 1 ml of diluted ECM solution into each well. Incubate the coated plate O/N at 37 °C. Wash the coated plate with pre-warmed DMEM/F12 medium before use.
3. Collect primary human hair from the individual’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 and determine the growth phase of the hair under a dissecting microscope (Figure 1).
   1. Collect 5-10 anagen hairs from each individual to extract keratinocytes. Process plucked hairs as soon as possible to ensure successful derivation of keratinocytes.
      Note: Figure 1 illustrates the morphology of hairs in different phases of the growth cycle. Anagen hair is in the growth phase, with a visible outer layer of epithelium surrounding the length of the hair, known as the ORS. On the other hand, telogen hair represents hair in the resting phase and does not have a visible ORS, but has a clear ball of cells covering the root of the hair, known as the telogen bulge.
4. Place the hair samples into a petri dish containing 10 ml of Antibiotic Mix to wash for 5 min at RT.
5. Using sterilized scissors and forceps, cut off excessive hair shaft, leaving a hair fragment with an intact hair follicle and around 0.5 – 1 cm of hair shaft.
6. Carefully transfer 1 hair fragment into each well of the ECM-coated plate using forceps.
7. Using a 1 ml pipette, add approximately 3 drops of Knockout Serum Replacement (KSR) medium (~100 – 200 µl) to the hair samples.
   Note: Keep the hair fragments in close contact to the plate surface to allow for attachment and subsequent keratinocyte outgrowth. Adding excessive media at this step may causes the hair fragments to float and decreases the chance of successful hair attachment.
8. Incubate the hair samples in a 37 °C incubator with 5% CO₂ and allow the hair follicles to attach O/N. On the following day, gently add another 3 drops of KSR medium (~100 – 200 µl) to keep the hair samples moist.
9. Observe the hair samples daily to confirm that the hair follicles have attached successfully. Generally attachment of hair follicles is apparent within 1-3 days. Thick hairs are usually easier to attach compared to fine hairs, as fine hairs have higher tendency to float which hinder the attachment process.
   1. Once the hair have attached to the coated plate, add 500 µl of KSR medium to the well. Take extra care when adding the medium as hair samples may not have attached firmly. From this point, change media every 2 days.
      Note: Keratinocyte outgrowths should be visible from the ORS region of the hair fragment after 3-7 days (Figure 2A-B). Allow the keratinocytes to grow up to 14 days before passaging the cells as described in the next section.

2. Maintenance and Passaging of Keratinocytes

1. Pre-coat a 6-well plate using the Coating Matrix Kit. Add 680 µl of the dilution medium from the kit to each well, followed by 6.8 µl of Coating Matrix solution. Shake the plate to ensure uniform distribution of the Coating Matrix solution on the plate.
2. Allow the coated plate to incubate for 30 min at RT. Remove the Coating Matrix solution prior to plating of keratinocytes.
3. Dissociate keratinocytes into a single cell suspension with 500 µl of 0.25% trypsin per well for 2-5 min at 37 °C. Inactivate the trypsin with 500 µl of medium containing serum (e.g., Fetal Bovine Serum (FBS) medium) and collect into a sterile 15 ml tube. Remove the hair fragment using sterilized forceps.
4. Determine the number of harvested cells using a haemocytometer.
5. Centrifuge the cells for 3 min at 200 x g. Wash the cells with PBS and repeat centrifugation. Aspirate the PBS and resuspend the cell pellet in fresh Keratinocyte medium.
6. Plate down 4 × 10⁴ keratinocytes into one well of ECM-coated plate in the presence of Keratinocyte medium. Keep the keratinocytes in a 37 °C incubator with 5% CO₂ (Figure 2C). Change media every day and passage cells when the culture is ~80% confluent, which may take approximately 2-7 days depending on the passage number and proliferation rate of the keratinocytes. The derived keratinocytes should be expanded to generate sufficient frozen stocks using standard slow-freezing cryopreservation methods¹⁶.
   Note: The described culture conditions support the expansion of homogenous population of keratinocytes with typical cobblestone morphology (Figure 2C). Occasionally, a few differentiated keratinocytes, with large and flat morphology and have lost replicative potential, may be observed in culture. Characterization of keratinocytes can be performed by immunostaining using antibodies against cytokeratin 14 as previously described ¹⁷. Established keratinocyte cell lines should also be tested for mycoplasma after 1-3 passages using commercially available kit.

3. Generation of hiPSCs from Keratinocytes Using Episomal Vectors

1. Pre-coat a 6-well plate with 0.1% gelatin (2 ml/well). Allow gelatin to coat for at least 20 min at RT.
2. For reprogramming experiment, use keratinocytes no later than passage 6. Dissociate keratinocytes into a single cell suspension with 1 ml of 0.25% trypsin per well for 2-5 min at 37 °C. Inactivate trypsin with 1 ml of media containing serum (e.g., FBS media) and collect in a 15 ml tube.
3. Centrifuge cells for 3 min at 200 x g and resuspend cells in Keratinocyte medium. Plate 1 × 10⁵-1.5 × 10⁵ keratinocytes per well in a gelatinized plate O/N.
4. On the following day (Day 0), check the plate to ensure cells are 50-60% confluent.
   Note: If keratinocytes are less than ~50% confluent they might not grow well after transfection.
5. On Day 0, perform transfection of the episomal reprogramming vectors on the keratinocytes, using a ratio of 8 µl transfection reagent to 2 µg total plasmid DNA.
   1. Take 0.5 µg for each of the 4 plasmids (pCXLE-eGFP, pCXLE-hOct3/4-shP53F, pCXLE-hSK, pCXLE-hUL) and dilute in 100 µl of Reduced Serum medium (e.g., OPTI-MEM).
      Note: The plasmid pCXLE-eGFP is used to monitor the transfection efficiencies in keratinocytes and is not required for successful reprogramming.
   2. Add 8 µl of transfection reagent to the diluted DNA. Mix by flicking the tube. Allow the DNA reaction mix to rest undisturbed at RT for 15 min.
   3. Replace the Keratinocyte medium with fresh medium.
   4. Gently add the DNA reaction mix to keratinocyte culture and incubate in a 37 °C incubator with 5% CO₂ for 4 hr.
      Note: It is not recommended to perform prolonged transfection O/N in keratinocytes as they exhibit low survival post-transfection.
   5. After 4 hr post-transfection, replace the Keratinocyte medium with fresh medium.
6. On Day 1 (1 day post-transfection), change the keratinocyte medium and check transfection efficiency by estimating the percentage of eGFP positive cells under a fluorescent microscope. Typically > 60-70% transfection efficiency is observed for primary keratinocytes using this protocol.
7. On Day 2, repeat transfection as described in step 3.5. Two consecutive transfections would typically achieve > 90% transfection efficiency. Keratinocyte culture usually reaches confluence by the end of the second transfection.
   1. Prepare a 10 cm dish of mouse embryonic fibroblast (MEF) feeder for each reprogramming reaction. Prepare mitotically inactivated MEF feeders as previously described ¹⁸.
   2. Pre-coat a 10 cm dish with 5 ml of 0.1% gelatin for at least 20 min. Plate 4 × 10⁶ mitotically inactivated MEF per 10 cm dish in FBS medium. Allow the cells to settle and attach O/N in a 37 °C incubator with 5% CO₂.
8. On Day 3, harvest the keratinocytes with 0.25% trypsin as described in step 3.2 and 3.3. Resuspend the keratinocytes in KSR medium and plate 90% of keratinocytes from 1 well of a 6-well plate into a 10 cm MEF feeder dish with KSR medium.
9. From Day 4 onwards, change the KSR medium every day.
   1. Alternatively after Day 18, replace culture medium every second day.
      Note: Non-reprogrammed keratinocytes will cease to proliferate, while hiPSC colonies with defined boundary that resemble human embryonic stem cells (hESCs) will emerge from Day 14 – 21.
10. Isolate hiPSC colonies by manual dissection from Day 21 onwards. Avoid hiPSC colonies that are closely clustered together and only pick hiPSC colonies that are clearly separated from others.
    Note: hiPSC colonies can be picked earlier,  but the colony with defined boundaries might not be obvious at earlier time points given the small colony size. Reprogramming efficiencies may vary between different patient’s keratinocytes, and is also influenced by other factors such as passage number or proliferation rate of keratinocytes.
    1. For manual dissection of hiPSC colonies, use a 26G needle to cut hiPSC colonies under a dissecting microscope. Cut a hiPSC colony into small pieces around 300 – 600 µm in length. Transfer the pieces from one hiPSC colony into a new MEF feeder plate (2.5 × 10⁴ MEF/cm²) to establish a clonal line of hiPSCs. Initially, culture each hiPSC clonal line in one well of a 12 well plate or organ culture dishes, then expand to 6 well plate format.
    2. Maintain established clonal lines of hiPSCs in KSR medium on MEF feeders. Once the culture reach 70 – 80% confluency with hiPSC colonies ~1.5 mm in diameter, passage hiPSCs using standard enzymatic passaging methods (Accutase, Dispase or Collagenase IV) according to the manufacturer’s instruction.
11. Characterize established hiPSC lines to confirm pluripotency and their ability to differentiate into cells representative of the three germ layers in vitro and/or in vivo^13,19. Also, routinely test established hiPSC lines for mycoplasma using commercially available kits to ensure they are pathogen-free. Routinely monitor genomic stability of hiPSC lines  by karyotyping or array-based copy number variation (CNV) analysis.