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

Summary

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Abstract

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

Introduction

The discovery of human induced pluripotent stem cells (hiPSCs) has revolutionized the field of regenerative medicine, providing a feasible method for generation of patient-specific stem cells ^1-3. hiPSCs have been successfully generated from various somatic cell types, including fibroblasts ^4,5, hematopoietic cells ^6,7, renal epithelial cells from urine ⁸ and keratinocytes ^9,10. To date, skin fibroblasts and hematopoietic cells represent the most commonly used cell sources for generating patient-specific iPSCs. Arguably, this is due to the fact that skin biopsies and blood collection are routine medical procedures and large biobanks of patient blood or skin samples have been established in many countries.

In contrast to blood cells and skin fibroblast which require invasive extraction methods, keratinocytes represent an easily accessible cell type for hiPSC generation. Keratinocytes are keratin-rich epithelial cells that form the exterior epidermal barrier of the skin and are also found in nails and hair ¹¹. In particular, keratinocytes can be found on the outer root sheath (ORS) of hair follicles, an external cellular layer that covered the hair shaft together with the inner root sheath (IRS) cells (¹², Figure 1). As hair collection is a simple procedure that does not require the assistance of medical personnel, it provides an opportunity for patients to collect and send their own hair samples to laboratories, which would greatly facilitate the collection of patient samples for hiPSC generation. Epidermal keratinocytes also have a higher reprogramming efficiency and faster reprogramming kinetics compared to fibroblasts, adding to the advantages of using keratinocytes as the starting cells for hiPSC generation ^9,13. Furthermore, hiPSCs can also be generated using other cell populations within the hair follicle, including the dermal papilla cells located at the base of the hair follicle ^14,15.

Previous reports of iPSC generation using hair-derived cells often utilize retroviral or lentiviral-based reprogramming methods ^9,14,15. However, these viral methods introduce undesirable genomic integration of foreign transgenes during reprogramming. In comparison, the use of episomal vectors represents a feasible, non-viral reprogramming method to generate integration-free iPSCs ⁴. We have previously developed a simple, cost-effective and non-viral method to efficiently reprogram keratinocyte into hiPSCs using episomal vectors ¹³. Here we provide a detailed protocol for the generation of keratinocyte-derived hiPSCs, including the derivation of keratinocytes from plucked hair, expansion and maintenance of the keratinocytes and subsequent reprogramming to generate hiPSCs.

Protocol

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.

Results

The hair goes through 3 different phases of growth cycle: anagen (the growth phase), catagen (the regression phase) and telogen (the rest phase) ^20,21. The anagen hair follicle contains multiple layers of epithelium; these layers include the ORS, IRS and the hair shaft (Figure 1). Anagen hair eventually undergoes transition to the catagen phase, which is marked by apoptosis of the ORS and termination of hair shaft differentiation. Finally, catagen hair transition to the telogen phase, where ap...

Discussion

Generation of patient-specific hiPSCs offers a unique approach for studying pathogenesis in the diseased cell types in vitro, and also provides a platform for drug screening to identify novel molecules that can rescue the disease phenotypes. This disease modeling approach using hiPSCs has yielded promising results for a variety of diseases, including Long QT syndrome, Huntington’s disease, Parkinson’s disease and amyotrophic lateral sclerosis ²². Several initiatives are already underway to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic Mix:
250 ng/ml Antimycotic amphotericin B	Sigma	A2942-20ml	Antibiotic mix is made up in PBS.
1X Penicillin/Streptomycin	Invitrogen	15140-122
PBS (-)	Invitrogen	14190-144
Knockout Serum Replacement (KSR) medium:			KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.
20% knockout serum replacement (KSR)	Invitrogen	10828-028
DMEM/F12 with glutamax	Invitrogen	10565-042
1× MEM non-essential amino acid	Invitrogen	11140-050
0.5× Penicillin/Streptomycin	Invitrogen	15140-122
0.1 mM β-mercaptoethanol	Invitrogen	21985
bFGF (10 ng/ml, added fresh)	Millipore	GF003
Keratinocyte medium:
EpiLife with 60 µM Calcium	Invitrogen	M-EPI-500-CA
1× Human keratinocyte growth supplement (HKGS)	Invitrogen	S-001-5
Fetal Bovine Serum (FBS) medium:			FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.
10% fetal bovine serum (FBS)	Invitrogen	26140079
DMEM	Invitrogen	11995-073
0.5x Penicillin/Streptomycin	Invitrogen	15140-122
2 mM L-glutamine	Invitrogen	25030
0.25% trypsin-EDTA	Invitrogen	25200-056
Extracellular Matrix (ECM):
Matrigel	Corning	354234	Aliquot Matrigel stock and store in -80°C following manufacturer’s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200µl matrigel for coating a 12-well plate (~150µg/well).
Coating Matrix Kit	Invitrogen	R-011-K
Plasmids:			Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.
-          pCXLE-eGFP	Addgene	27082
-          pCXLE-hOct3/4-shP53F	Addgene	27077
-          pCXLE-hSK	Addgene	27078
-          pCXLE-hUL	Addgene	27080
Transfection reagent Fugene HD	Promega	E231B
Gelatin (from porcine skin)	Sigma	G1890	Make up 0.1% gelatin in distilled water. Autoclave before use.
Reduced Serum medium: OPTI-MEM	Invitrogen	31985062
Accutase	Sigma	A6964-100ml
Mouse embryonic fibroblast (MEF) feeder			MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.
26G needle	Terumo	NN2613R
6-well plate (tissue culture treated)	BD Biosciences	353046
12-well plate (tissue culture treated)	BD Biosciences	353043
10 cm dish (tissue culture treated)	BD Biosciences	353003
Dispase	Invitrogen	17105-041	Use at 10 mg/ml
Collagenase IV	Invitrogen	17104-019	Use at 1 mg/ml
TRA-160 antibody	Millipore	MAB4360	Use at 5 µg/ml
OCT4 antibody	Santa Cruz	SC-5279	Use at 5 µg/ml
NANOG antibody	R&D Systems	AF1997	Use at 10 µg/ml
MycoAlert Detection kit	Lonza	LT07-418