protocol

Isolation and cultivation of human keratinocytes

from skin or plucked hair for the generation of
induced pluripotent stem cells
Trond Aasen1, 2 & Juan Carlos Izpisúa Belmonte1, 3
1
Center of Regenerative Medicine in Barcelona, Barcelona, Spain. 2Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Zaragoza, Spain. 3Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA. Correspondence should be addressed to
J.C.I.B. (belmonte@salk.edu and izpisua@cmrb.eu).
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 4 February 2010; doi:10.1038/nprot.2009.241

The ease of generating induced pluripotent stem (iPS) cells, and possibly their properties after reprogramming, depends on the
origin of the somatic cell starting population. Reprogramming of keratinocytes is both faster and more efficient compared with
fibroblasts, although more care is required when isolating, culturing and infecting these cells. In this study, we describe detailed
protocols using both feeder-dependent and defined serum- and feeder-free conditions for culturing human keratinocytes from
foreskin samples and punch biopsies, as well as how to isolate keratinocytes from plucked hair. We further describe culture
techniques and approaches to efficiently infect and reprogram these cells for the purpose of generating iPS cells. The procedure
of deriving keratinocytes takes 10–14 d, whereas reprogramming and the appearance of iPS cell colonies that can be isolated and
established requires another 3–4 weeks.

INTRODUCTION
Keratinocytes importance of avoiding any long-term exposure to high levels of
The epidermis is a thin nonvascular layer (Fig. 1) consisting mainly calcium, which effectively induces keratinocyte differentiation.
of stratifying epithelial keratinocytes. These cells undergo contin­ Using the classical serum- and feeder-based technique has other
uous and rapid proliferation and are thought to be continually advantages, including rapid proliferation and significantly higher
regenerated from a pool of multipotent stem cells1. As keratino­ resistance to apoptosis, e.g., after adenoviral infection. Compared
cytes leave the basal layer and move upward, they undergo ter­ with some cell types such as fibroblasts, keratinocytes tend to require
minal differentiation forming a continually shedding protective more care and issues such as apoptosis in low density and, particu­
barrier at the surface of the skin. Skin fibroblasts are situated in the larly, differentiation and senescence when reaching confluence are
thicker dermis (Fig. 1), which is situated directly below the epider­ more prominent. Although keratinocytes can undergo a relatively
mis (separated by the basement membrane), and provide, among few number of passages (at least under the classical culture condi­
other functions, growth factors for keratinocytes, a feature that has tions described here), they are a great source of highly proliferating
proved important for early keratinocyte culture models. Thus, it
has been possible for many years to efficiently isolate both keratino­
cytes and fibroblasts from the same skin sample. Several other cell
types can also be isolated from skin, including endothelial cells and
epidermal melanocytes. In addition, keratinocytes can be isolated
from plucked hair. The hair follicle (Fig. 1) is a complex epider­
mal appendage embedded deeply in the dermis, which continually
cycles between anagen (rapid growth), catagen (regression) and
Epidermis
telogen (resting) phases. Outer root sheath (ORS) cells surround
the hair follicle essentially as a stratified epithelium of keratinocytes
that is contiguous with the epidermis making them easy to isolate.
The hair follicles contain their own stem cells (notably in the bulge, Dermis
an area of the ORS), which in addition to generating ORS keratino­
Hair
cytes have the capacity to contribute to other cell types2 as well as follicle
to epidermal keratinocytes after wound healing3.
Fatty
tissue
Keratinocyte culture
Keratinocytes can be cultured based on the traditional feeder-
dependent method developed by Rheinwald and Green4 or by Figure 1 | Schematic of human skin. Human skin consists of three main
more recent methods using defined serum-free, low-calcium media parts: the hypodermis (fatty subcutaneous tissue), the dermis (collagenous
tissue from which fibroblasts can be isolated) and the epidermis (stratifying
that do not require feeder cells5; both strategies have advantages
epithelial tissue consisting mainly of keratinocytes and also other cell types
and disadvantages. For example, culturing cells in serum-free, including melanocytes). Human hair is a complex mini-organ of which
low-calcium conditions avoids the need of feeder cells and rapidly keratinocytes in the outer root sheath are differentiating inward to produce
removes contamination of other cells such as fibroblasts, as they do the keratin of the growing hair. These cells can be isolated for culture by
not grow well in these conditions. One disadvantage is the crucial plucking or dissecting the hair.

nature protocols | VOL.5 NO.2 | 2010 | 371

 protocol
epithelial cells and have proved invaluable for many cell biology
studies, including the development of complex three-dimensional
tissue models and for grafting in wound therapy such as in severe
burn patients.
Plucked hair ORS keratinocytes can be obtained in culture
by various ways6, either by direct outgrowth on tissue culture
plastic7,8 or by enzymatic digestion9. In addition, there are a number
of reports describing the isolation of many multipotent stem cells
both in mouse and in human2,10–13. However, it should be noted
that direct plucking of hair (as described here) will isolate mainly
transit-amplifying cells with short-term culture potential, whereas
tissue microdissection enables isolation of the complete bulge and
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols
dermal papilla and the associated stem cells.
Keratinocytes and iPS cell technology
During recent years, reprogramming of somatic cells into the
so-called induced pluripotent stem (iPS) cells has been shown by
forced expression of certain trancription factors14,15, notably the
combination of Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells are
highly similar to embryonic stem (ES) cells and can be characterized
by the unlimited self-renewal potential and the ability to differenti­
ate into any cell type. Thus, the generation of iPS cells has not only
revolutionized the field investigating the molecular mechanisms of
cellular pluripotency but also facilitated the generation of patient-
specific cells for cell replacement therapy16. In addition, ethical and
host-rejection issues associated with classical ES cell technology
have been reduced, spawning enormous interest and promise for
future clinical applications. However, the process of reprogramming
is slow and inefficient, and the full extent of whether iPS cells can
replace ES cells in every aspect is still being debated, and partial
reprogramming or ‘over-reprogramming’ poses challenges15.
It is thought that the process of iPS cell generation depends on
many factors, including age, type and origin of the cells used. Thus,
it is still unclear what the ultimate source of cells will be with regard
to both availability and applicability after reprogramming and
re-differentiation.
As keratinocytes (unlike fibroblasts) are epithelial cells, contain
putative stem cells and are highly accessible, we recently explored
MATERIALS
REAGENTS
• EpiLife Medium with 60 µM calcium (Invitrogen, cat. no. M-EPI-500-CA)
• EpiLife Human Keratinocyte Growth Supplement (HKGS) (Invitrogen,
cat. no. S-001-5)
• Coating matrix (Invitrogen, cat. no. R-011-K)
• Phosphate-buffered saline (PBS; e.g., Invitrogen, cat. no. 10010-056)
• Hanks’ balanced salt solution (HBSS) without Ca2 +  and Mg2 +  (Invitrogen,
cat. no. 14170-088)
• Penicillin/streptomycin (Invitrogen, cat. no. 15140-122)
• Antimycotic amphotericin B solution (Sigma, cat. no. A2942-20ml)
• Dispase (Becton Dickinson, S.A., cat. no. 354235)
• 0.25% (wt/vol) Trypsin/EDTA (Invitrogen, cat. no. 25200-056)
• 0.05% (wt/vol) Trypsin/EDTA (Invitrogen, cat. no. 25300-054)
• TrypLE Express Stable Trypsin Replacement (Invitrogen,
cat. no. 12604-039)
• Matrigel (Becton Dickinson, S.A., cat. no. 356234) (see REAGENT SETUP)
• DMEM (Invitrogen, cat. no. 11965-092)
• Knockout (KO) DMEM (Invitrogen, cat. no. 10829-018)
• DMEM/F12 (Invitrogen, cat. no. 11330-32)
• Heat-inactivated FBS (Invitrogen, cat. no. 10270-106)
• KO Serum Replacement (KOSR; Invitrogen, cat. no. 10828-028)
• GlutaMAX (Invitrogen, cat. no. 35050-038)
372 | VOL.5 NO.2 | 2010 | nature protocols
the possibility of using ectodermal skin keratinocytes as a starting
population for iPS cell generation. Our study showed that repro­
gramming of these cells was at least 2-fold faster and 100-fold
more efficient compared with fibroblasts17. We have also shown the
applicability of culturing keratinocytes using punch biopsies from
Fanconi anemia patients. Lentiviral expression of a healthy gene,
in order to correct the phenotypic defect, allowed the generation
of keratinocyte iPS (KiPS) cells and re-differentiation of these cells
to the blood lineage18. In addition, we have shown that keratino­
cytes can be cultured and reprogrammed from single plucked hair17,
thus offering a simple and accessible route to KiPS cell generation,
avoiding the need of medical personnel and with minimum incon­
venience for the patients.
In this study, we describe detailed protocols to isolate and culture
keratinocytes from both skin biopsies and plucked hair, and explain
how to efficiently infect these cells in order to generate iPS cells. For
both epidermal and plucked hair keratinocytes, we describe the two
major approaches to culture: serum/feeder-based (faster growth,
bulk production) and serum-free culture (slower growth, more
undifferentiated). Both models are likely to be important depend­
ing on the individual approaches and studies by different labora­
tories. One aspect that is important to be aware of, particularly if
using serum-free formulations to culture keratinocyte, is their high
sensitivity to calcium and contact-induced differentiation. Calcium
and high cell density are potent inducers of differentiation in
keratinocytes, which should be taken into consideration both during
culture and reprogramming. Cultured keratinocytes should also
be amenable to a vast array of other applications including repro­
gramming using non-integrative strategies. The protocols can also
serve as an experimental platform for studying pluripotency and
reprogramming, e.g., to compare changes that occur in mesenchy­
mal versus ectodermal cells during reprogramming or the isolation
and reprogramming of a specific keratinocyte population includ­
ing putative stem cells. With respect to such comparative iPS cell
technology, the use of a small skin biopsy is particularly useful, as
mesenchymal fibroblasts14, ectodermal keratinocytes17 and, more
recently, neural crest-derived melanocytes19 can all be cultured and
reprogrammed into iPS cells.
• Non-essential amino acid solution (Invitrogen, cat. no. 11140-050)
• 2-Mercaptoethanol (Sigma, cat. no. M7522) ! CAUTION Toxic by inhalation
and skin contact; wear gloves and work in a tissue culture hood.
• bFGF, human recombinant, 1,000 µg stored at  − 20 °C (Peprotech,
cat. no. 100-18B) (see REAGENT SETUP)
• Human serum albumin (HSA) solution (100 mg ml − 1, VitroLife/EMB,
cat. no. 10064)
• PBS without calcium and magnesium (Invitrogen, cat. no. 2531)
• SARSTEDT tubes (1.5 ml) (Sarstedt, cat. no. 72.692.005)
• Gelatin 0.1% (wt/vol) solution (Millipore, cat. no. ES-006-B)
• Dimethyl sulfoxide (DMSO; Sigma, cat. no. D4540)
• FuGENE 6 transfection reagent (Roche Applied Science,
cat. no. 1181509001)
• Cholera toxin (2 mg; Sigma, cat. no. C8052) ! CAUTION Highly toxic when
inhaled. Wear gloves and work in a tissue culture hood. It may require
licensing every time it is ordered, which may take several months.
• Epidermal growth factor (EGF, recombinant, human, 2 mg) (Sigma,
cat. no. E9644-2MG)
• Hydrocortisone (Sigma, cat. no. H4881)
• Insulin (Sigma, cat. no. I5500)
• Transferrin (500 mg; Sigma, cat. no. T2252)

• 3,3,5-Triiodo-L-thyronine sodium salt (100 mg; Sigma, cat. no. T6397)
• Polybrene (10 mg ml − 1; Chemicon, cat. no. TR-1003-6)
• Mouse embryo fibroblast (MEF), human fibroblast and 293 Phoenix
Ampho cell medium (see REAGENT SETUP)
• RM +  keratinocyte medium (see REAGENT SETUP)
• hES cell medium (see REAGENT SETUP)
• Freezing medium (see REAGENT SETUP)
• pMSCV retroviral vectors or similar, containing cDNA for expression of
Oct4, Sox2, Klf4, c-Myc and GFP (see REAGENT SETUP)
• 293 Phoenix Ampho cells (Orbigen, cat no. RVC-10001) human embryo
kidney, epithelial (ATCC, SD-3443)
• MEFs: For preparation and use, see previous Nature Protocols20
• Swiss 3T3-J2 mouse fibroblasts (originally derived by Dr. Howard Green,
Harvard Medical School, Boston, MA) or human fibroblast feeder cells.
• Source of keratinocytes (see REAGENT SETUP): foreskin sample, 2- to
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols
6-mm skin punch biopsy or plucked hair. Alternatively, keratinocytes
may be purchased commercially (e.g., Invitrogen, cat. no. C-001-5C)
! CAUTION Tissue samples must be obtained by an appropriately
trained physician with informed consent and under a protocol approved
by the appropriate national and host institutional boards and ethical
committees.
EQUIPMENT
• Biosafety cabinet with aspirator for tissue culture
• Tissue culture dishes, 12- and 6-well, and 60, 100 and 150 mm
• Petri dishes: 100 mm
• Incubator: 37 °C, 90% humidity, 5% CO2
• Water bath: 37 °C
• Cell counter or hemocytometer
• Pipettes
• Centrifuge
• Inverse phase-contrast microscope
• Stereomicroscope
• 500-ml bottle filter
• Scalpel
• Tweezers
• Scissors
• Cell strainer (70 µm, Becton Dickinson, cat. no. 352350)
• Disposable sterile filter system (0.22 µm, 500 ml; Corning, cat. no. 430758)
• Filter (for retrovirus), Millex-HV PVDF 0.45 µm (Millipore, cat. no.
SLHV033RS)
• 15- and 50-ml Falcon tubes
• Cryovials (Sigma, cat. no. V7634-500EA)
• Freezing container (Nalgene Labware, cat. no. 5100)
REAGENT SETUP
HBSS with antibiotics  Add 1% (vol/vol) penicillin/streptomycin antibiotics
and 1 µl of 10 ml amphotericin B solution to reach final concentrations of
100 U ml − 1 penicillin, 100 mg ml − 1 streptomycin and 250 ng ml − 1 amphotericin B.
Store at 4 °C for up to 2 weeks.
HBSS with antibiotics and dispase  Add 1 ml dispase solution
(5,000 caseinolytic units) to 9 ml HBSS with antibiotics. Store at 4 °C for
up to 2 weeks.
EpiLife medium  Defrost a 5-ml vial of EpiLife HKGS and add to a 500-ml
bottle of EpiLife medium. HKGS contains a mixture of bovine pituitary
extract, bovine insulin, bovine transferrin, hydrocortisone and human EGF
(details can be found at Cascade Biologics homepage). Add 5 ml antibiotics
to final concentrations of 100 U ml − 1 penicillin, 100 mg ml − 1 streptomycin
and add 50 µl amphotericin B for a final concentration of 250 ng ml − 1. At
later passages, remove the antibiotics if it is important to achieve optimum
growth conditions.  CRITICAL Store in the dark at 4 °C and use within 2
weeks.  CRITICAL Calcium concentration must not be higher than 60 µM.
 CRITICAL Other serum-free keratinocyte medium formulations that sup­
port growth equally well may display lower levels of infection or transgene
expression levels.
RM +  supplement, individual stock solutions (keep at  − 20 °C)  The
individual stocks required are transferrin (50 mg ml − 1, dissolved in ddH2O),
hydrocortisone (4 mg ml − 1, dissolved ddH2O), cholera toxin (0.1 mg per
10 ml, dissolved in ddH2O), EGF (0.1 mg ml − 1 dissolved ddH2O) and
insulin (50 mg ml − 1 dissolved 0.05 M of HCl solution). To make up liothyronine
stock, first add 6 mg (MW 651.0) liothyronine (3,3,5-triiodo-l-thyronine
sodium salt) to 200 µl 1 N NaOH solution with constant swirling and
protocol
then add 9.8 ml ddH2O. Aliquot into 0.5 ml per vial (L1; concentration
9.2 × 10 − 4 M). Next, take one 0.5 ml vial of L1, add 12.5 ml ddH2O and pre­
pare aliquots (1 ml per vial) (L2; concentration 3.5 × 10 − 5 M). Add 1 ml of
L2 to 9 ml ddH2O (total volume 10 ml) and prepare aliquots (1 ml per vial)
(L3; concentration 3.5 × 10 − 6 M). Finally, aliquot 1 ml of L3 (100 µl per vial)
(1.77 × 10 − 9 M). ! CAUTION Cholera toxin is highly toxic.
100× RM +  stock (200 ml)  Start by slowly re-suspending the insulin in
ddH2O. To do this, pipette the 2 ml vial along with 5 ml of ddH2O against
the measuring cylinder as slowly as possible. In any order, add 200 µl of
liothyronine and 2 ml of all the other chemicals. Top up the cylinder until
it reaches 100 ml, filter and then add 100 ml of DMEM/F12 medium to the
cylinder. Filter through a 20-µm filter (total volume 200 ml). Final 100×
concentrations will be transferrin 500 µg ml − 1, hydrocortisone 40 µg ml − 1,
cholera toxin 1 × 10 − 8 M (final concentration may differ by 19%, but this is
acceptable), EGF 1 µg ml − 1, insulin 500 µg ml − 1, liothyronine 1.77 × 10 − 9 M.
Aliquot into 5 ml and keep at  − 20 °C for up to 1 year.
RM +  medium  A modified keratinocyte medium was prepared by
Gilfix and Green21.
Prepare a 1:1 mixture of DMEM and DMEM/F12 medium (final mix will
be 3:1 DMEM/F12) containing 10% fetal bovine serum (FBS) (vol/vol), 1%
GlutaMAX (vol/vol), 50 U ml − 1 penicillin and 50 mg ml − 1 streptomycin, 1%
RM +  supplement (vol/vol) giving a final concentration of mitogens of
5 µg ml − 1 transferrin, 0.4 µg ml − 1 hydrocortisone, 10 − 10 M cholera toxin,
10 ng ml − 1 EGF, 5 µg ml − 1 insulin and 2 × 10 − 11 M liothyronine. Store at
4 °C for up to 2 weeks.  CRITICAL FBS batch variations can significantly
influence keratinocyte culture. Batch testing is recommended.
Matrigel preparation and coating  Thaw Matrigel on ice overnight at 4 °C.
Keep everything on ice until the dilution is prepared. When preparing the
dilution, use pre-cooled pipettes, tubes and media (not having everything cold
will cause the Matrigel to gel rapidly and this will result in clumps in the
solution). Prepare a 1:15 dilution of Matrigel in cold KO-DMEM without
any supplement (140 ml KO-DMEM  +  10 ml Matrigel) on ice. Mix it well,
aliquot and store at 4 °C for up to 1 month. Coating: place sufficient Matrigel
on tissue culture plates to cover the bottom, place overnight at 4 °C. Aspirate
and wash once with KO-DMEM before use.
Keratinocyte cell-freezing medium  Mix 1 part DMSO with 9 parts FBS.
Keep at 4 °C for up to 24 h. Use as described in Box 3.
Complete DMEM media for 293 Phoenix Ampho cells, MEFs or human
fibroblasts  High-glucose DMEM, 10% FBS (vol/vol), GlutaMAX 2 mM,
penicillin/streptomycin (100 U ml − 1 and 100 µg ml − 1, respectively). To
prepare 500 ml of the medium, mix 50 ml FBS, 5 ml GlutaMAX and 5 ml
penicillin/streptomycin, and then fill up to 500 ml with DMEM. It can be
stored at 4 °C for 2–3 weeks.
bFGF  Centrifuge the liophilized bFGF for 1–2 min at 10,000 r.p.m. so that
all the content settles to the bottom. Prepare a 0.2% (wt/vol) HSA solution in
a sterile tube by adding 0.2 ml HSA solution (100 mg ml−1) to 9.8 ml of PBS.
Dissolve bFGF with PBS in 0.2% HSA to obtain a final concentration of
100 µg ml − 1. Prepare 50 µl aliquots in SARSTEDT tubes. Store at  − 20 °C
or  − 80 °C for up to 1 month.
hES cell medium  KO-DMEM containing 20% KOSR (vol/vol), 10 ng ml − 1
bFGF, 1 mM GlutaMAX, 100 µM Non-Essential Amino Acids, 100 µM
2-mercaptoethanol, 50 U ml − 1 penicillin and 50 mg ml − 1 streptomycin.
Filter the medium with a bottle-top 0.22-µm filter and store at 4 °C for
up to 1 week.
Gelatin-coated culture dishes  Add a 0.1% gelatin solution to cover the
bottom of the dish. Incubate the dish for at least 30 min at 37 °C. Aspirate
and allow to dry for at least 10 min in the tissue culture hood.
Feeder-conditioned media  Depending on the experiment, induce senes­
cence in either human foreskin fibroblasts (HFFs), MEFs or mouse 3T3-J2
fibroblasts by γ-irradiation of 55 Gy. In gelatin-coated 100-mm dish, seed
out 4 × 106 irradiated cells in complete DMEM medium. Place the medium
overnight in a 37 °C, 5% CO2 incubator. The next day, replace with 10 ml
medium (either RM +  or hES). Everyday, for the next 10 d, collect the medium
(replace with fresh medium), filter through a 0.22-µm filter and store the
filtrate at 4 °C for up to 1 week.
Retroviral vectors  pMSCV retroviral vectors containing Oct4, Sox2, Klf4
and c-Myc, available from Addgene (e.g., 20072, 20073, 20074 and 20075,
respectively).
nature protocols | VOL.5 NO.2 | 2010 | 373
 protocol

PROCEDURE
Isolation of keratinocytes from foreskin samples or punch biopsies ● TIMING 2 d
1| Place skin tissues in cold HBSS (or EpiLife medium) containing antibiotics and antimycotics and keep at 4 °C until use.
 CRITICAL STEP Samples should be processed as early as possible, ideally the same day or the next morning owing to
gradual loss of yield (particularly after 24 h). If it is a small biopsy sample ( < 5 mm), it may be advantageous to culture
the cells using explant outgrowth as described in Box 1.

2| Place the tissues in an uncoated 100-mm bacterial Petri dish and keep moist with some medium. Remove subcutaneous
fat and loose connective tissues (hypodermis) using fine tweezers and a scalpel. Open up or flatten the skin and place the
epidermis side down. Use the edge of the scalpel to scrape away tissues until only the thin epidermis and the dense dermis
remain (Fig. 2a). With foreskin samples, cut the piece into strips about 3–4 mm width (Fig. 2b).
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

 CRITICAL STEP It is advisable to perform this part of the procedure late in the afternoon if overnight incubation is
performed.

3| Place the pieces with the dermal side down in a 60-mm dish containing 5–6 ml of HBSS (or EpiLife) with antibiotics and
dispase (Fig. 2b). Cover and store the pieces in a sterile place at 4 °C for 12–16 h (overnight).

4| Take the overnight digested tissue pieces and grab the edge of the dermal part of the tissue with one tweezer and the
thin epidermal part with another set of thin tweezers and slowly peel off the epidermis (Fig. 2c). Immediately transfer
the epidermis (almost transparent) into another dish with either HBSS or EpiLife medium (Fig. 2d). If desired, preserve
the dermis (thicker and gooey) for fibroblast isolation as described in Box 2.
? TROUBLESHOOTING

5| Cut the epidermis using a scalpel into small pieces of 1 mm2.

6| Place the minced tissue pieces in a Falcon tube containing TrypLE Select. For a whole piece of foreskin, use a 50-ml
Falcon tube and about 20 ml TrypLE Select. For smaller samples, use a smaller-sized tube and a lesser amount of reagent.

7| Incubate at 37 °C for 40–45 min when using a whole foreskin (20–25 min for a small biopsy). Mix the sample gently
every 5 min. The solution should become turbid.
? TROUBLESHOOTING

Box
  1 | CULTURE OF KERATINOCYTES USING SERUM AND FEEDER CELLS
● TIMING 1–2 weeks
In some circumstances, it is preferential to culture cells using the traditional cell culture technique devised by Rheinwald and Green4.
For example, we have observed that adenoviral infection of keratinocytes grown in the serum-free system leads to rapid apoptosis,
whereas in serum/feeder conditions, efficient infection with cell survival is achieved. In addition, a higher level of proliferation is
observed and retention of the expression of keratinocyte markers is more consistent. At any point, it is possible to directly switch to
EpiLife medium if required. Switching from EpiLife to serum-based medium, however, is time-consuming and not recommended.
1. Isolate keratinocytes according to Steps 1–12 of the main protocol.
2. Pre-seed tissue culture plates with irradiated human fibroblasts (2 × 104 feeder cells per cm2 or about 1.2 × 106 cells per 100-mm dish).
 CRITICAL STEP For optimum culture of keratinocytes, it is may be advantageous to use mouse 3T3-J2 fibroblasts (originally derived
by Dr. Howard Green) as feeder cells. Irradiated MEFs can also be used.
3. Seed 2.5 × 106 keratinocytes to each 100-mm culture plate.
4. Carefully return the plate to the incubator.
5. Replace with fresh RM +  medium every 3–4 d. Within 10 d, clearly demarcated colonies of keratinocytes should be apparent (Fig. 2b).
6. When cells are 70–75% confluent, after about 2 weeks, aspirate the media and wash two times with PBS. Add 4–5 ml 0.05%
Trypsin/EDTA solution and incubate at 37 °C for 1 min to detach feeder cells.
7. Remove feeders by aspiration and add 4–5 ml of 0.25% Trypsin/EDTA, incubate for 5–8 min at 37 °C until keratinocyte colonies have
rounded up and some of the cells are floating.
8. Tap the culture dish firmly on the side to release at least 95% of the cells.
 CRITICAL STEP Ensure that most cells are detached from the dish. Perform a second round of trypsinization if necessary.
9. Add 10 ml of fresh RM +  medium and transfer to a 15-ml Falcon tube.
10. Centrifuge at 200g for 5 min.
11. Passage at a ratio of 1:4 or 1:5 in plates pre-seeded with irradiated feeder cells.
12. Change the medium every 3 d and split the cells before reaching 90% confluence (typically once every week). For freezing cells,
see Box 3.

374 | VOL.5 NO.2 | 2010 | nature protocols

 protocol

8| Add 20–30 ml of the medium containing a minimum of a b

10% serum (vol/vol) (e.g., DMEM or RM + ) and pipette the
solution vigorously up and down for 10–15 times.

9| Pass the solution through a 70-µm mesh filter into a

new Falcon tube to remove undigested pieces of tissue.

10| Centrifuge at 200g for 5 min. c d

11| Remove the supernatant and resuspend in 5 ml EpiLife
medium.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

 CRITICAL STEP If a feeder/serum-based medium is

required, resuspend in the RM +  medium and continue as
described in Box 1.

12| Count the number of cells (e.g., with a hemocytometer).
Serum- and feeder-free culture of keratinocytes
● TIMING 2–3 weeks
13| Coat the culture plates with Coating Matrix (type I
collagen) by adding ~5 ml of a coating solution to each
100-mm tissue culture plates and incubate for 30 min at
22 °C. The coated dishes may be stored at 4 °C for
several days.
 CRITICAL STEP Coating of the culture plates is important for maximum yield with initial seeding of cells but is not
essential during subsequent passages.
Figure 2 | Enzymatic isolation of the epidermis and the dermis. (a) Foreskin
or other skin sample is placed epidermal side down (in this case, dark
pigmented epidermis) and loose connective tissue is scraped away using a
scalpel. (b) The tissue is cut into smaller pieces of 4–5 mm width and placed
in dispase solution overnight at 4 °C. (c) The next day, the epidermis is
peeled off and placed in a second dish in the medium. (d) The end result is
one dish with the dermis that can be used for fibroblast isolation and the
other dish with the epidermis that can be used for keratinocyte isolation.

Box
  2 | ISOLATION OF FIBROBLASTS (IN ADDITION TO KERATINOCYTES) FROM
THE SAME SAMPLE ● TIMING 2–4 WEEKS
In biopsies and foreskin samples, but not plucked hair, a substantial amount of fibroblasts are found in the dermal part of the skin,
which may be cultured in addition to keratinocytes.
Procedure
1. After overnight digestion with dispase and removal of the epidermis (Steps 1–4), the dermis is placed in DMEM culture medium and
kept at 4 °C until use.
 PAUSE POINT Although not recommended, fibroblast can survive a long time under these conditions and it is possible to obtain
cells up to several days later.
2. Using sterile forceps and scalpels, cut the dermis into 0.5- to 1.0-mm pieces.
3. Using tweezers, dip each piece in DMEM culture medium and place directly in tissue culture plates. Place ~10 to 15 pieces in each
100-mm dish or use 60-mm dishes while obtaining cells from a biopsy rather than a foreskin sample.
4. Place 1–2 drops of complete DMEM medium onto each piece of tissue.
5. Incubate in a 37 °C, 5% CO2, 90% humidity incubator for a minimum of 4 h up to overnight (maximum).
 CRITICAL STEP Do not allow pieces to dry out completely.
6. Gently add 7–8 ml of complete DMEM medium to each 100-mm plate.
 CRITICAL STEP It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated in a new dish
following the two steps above.
? TROUBLESHOOTING
7. Carefully return the plate to the incubator.
8. Replace with fresh medium every 3–4 d and remove any tissue pieces that are floating.
9. Within 7–10 d, outgrowths of fibroblast should appear.
10. After 14–21 d, aspirate the medium and wash twice with PBS. Add 4–5 ml of a 0.05% Trypsin/EDTA solution and incubate at 37 °C
for 4–5 min.
11. Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells
and then immediately add 10 ml of complete DMEM medium.
12. Centrifuge at 200g for 5 min.
13. Passage at a ratio of 1:4 in 150-mm tissue culture dishes by changing the medium every 3 d and splitting cells before reaching
100% confluence (typically once every week). Use or freeze down as described in Box 4.

nature protocols | VOL.5 NO.2 | 2010 | 375

 protocol
Figure 3 | Human keratinocytes in culture.
(a) Human keratinocytes at passage 1 after a b c
cryopreservation growing in low-calcium serum-
free medium (EpiLife). Under these conditions,
cells are less adhesive and do not form tight
keratinocyte colonies. Note the presence of a few
very large and flat cells, which are differentiated
keratinocytes that have lost replicative potential.
(b) An example of a keratinocyte colony
growing in serum with surrounding feeder cells.
Keratinocytes are tightly packed and adhesive. (c) Using immunofluorescence, keratinocytes stained positive for keratin 14 (K14), a useful marker to identify
the cell population as basal keratinocytes. The nucleus is stained blue using DAPI, showing the absence of any other K14-negative cell type.
Scale bars represent 250 µm (a–b); 50 µm (c).
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

14| Remove the Coating Matrix and seed out about 2.5 × 106 cells (4 × 104 cells per cm2) in ~10 ml EpiLife medium for each
100-mm tissue culture plate.

15| Change the medium the next day and subsequently for every 3 d, paying close attention to cell density. Under these
culture conditions, it may take several days before healthy colonies of cells are visible (Fig. 3).

16| When cells reach 70–75% confluence, rinse with PBS (without calcium) and trypsinize cells using ~4 ml of TrypLE Select
for each 100-mm plate and incubate at 37 °C. Cells round up and should come off the plate within 4–5 min. Gently tap the
dish to release the cells.
 CRITICAL STEP Do not allow cells to become confluent so as to avoid differentiation and reduced viability.

17| Add ≥10 ml of EpiLife and centrifuge cells at 200g for 5 min.

18| At this point, continue with subsequent passages with a split ratio of about 1:4. Alternatively, freeze-down the cells
(as described in Box 3), or use the cells immediately for reprogramming into KiPS (Steps 38–48). After this point, the use
of Coating Matrix is not essential and in our experience only marginally increases attachment and growth.

Culture of keratinocytes from plucked hair ● TIMING 1–2 weeks

19| Prepare a non-coated 100-mm bacterial plate containing HBSS and antibiotics.

20| Use tweezers to gently pull the hair out and place in HBSS medium. It is possible to use hair from any part of the body,
although the occipital part of the head is particularly suitable and accessible, giving many hairs in anagen growth phase
with a large amount of ORS cells on the hair shaft (Fig. 4a).
 CRITICAL STEP Although a noninvasive procedure, the donor should agree and sign all ethical guidelines as with normal biopsies.
 CRITICAL STEP Hair should be in anagen growth phase with a clearly visible ORS (Fig. 3a) and should be immediately
transferred to some medium to avoid the drying out of the cells on the hair.

21| While submerged in the medium, cut off the external part of the hair leaving the bulb and ORS (one may also remove
the bulb of the hair).

22| At this stage, two optional procedures for growing keratinocytes from the plucked hair is described, direct outgrowth
(option A) and enzymatic digestion (option B), which may be chosen depending on the scale of the experiment and
subsequent assay to be performed by the researcher. Option A was adopted for use with HFF- or MEF-conditioned hES
medium for subsequent direct reprogramming, although standard HFF or MEF-conditioned RM +  medium may also be used.
Option B allows for isolation of single cells in a defined medium.
(A) Direct outgrowth of keratinocytes from plucked hair (for direct iPS generation) ● TIMING 10–14 d
(i) Coat the required number of 35-mm culture dishes with Matrigel by adding sufficient Matrigel to cover the plate and
incubate overnight at 4 °C.
(ii) Place the hair obtained from Step 20 in the coated culture plates.
(iii) Gently add some drops of MEF-conditioned hES medium (or MEF-conditioned RM +  medium) sufficient to keep the hair
moist (see REAGENT SETUP).
 CRITICAL STEP During the first 24 h, it is absolute essential to ensure that the hair sticks to the culture dish and
does not float.
? TROUBLESHOOTING

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 protocol

Box
  3 | FREEZING AND THAWING KERATINOCYTES, FIBROBLASTS AND PHOENIX
293 CELLS ● TIMING 1–2 H
All primary cells described in this protocol can be frozen and stored indefinitely after isolation. We follow the exact same procedure
with all cells while using the recommended medium for each cell type.
Procedure for freezing cells
1. Ensure that the cells are maintained in exponential growth phase.
2. When cells reach 75–90% confluence, wash twice with PBS and add
  (i) 3–4 ml of TrypLE for keratinocytes grown in EpiLife;
  (ii) 3–4 ml of 0.05% Trypsin/EDTA for fibroblasts and Phoenix Ampho 293 cells; and
  (iii) 3–4 ml of 0.25% Trypsin/EDTA for keratinocytes grown in serum with feeders.
 CRITICAL STEP Remove feeder cells, if present, by incubating for 1 min with 0.05% Trypsin/EDTA.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

3. When cells have rounded up and some have detached, tap the tissue culture plate firmly on the side to detach the rest of the
cells.
4. Add 10 ml culture medium corresponding to cell type and pipette up and down in the dish four to five times.
5. Take 10 µl of the medium for counting the cells while performing next the step.
6. Centrifuge at 200g for 5 min.
7. Resuspend the pellet in 4 °C freezing medium such that a density of 1 × 107 cells per ml is obtained.
8. Aliquot 1 ml into each cryovial and place in a ‘Mr Frosty’ freezing unit that has been pre-cooled to 4 °C. Place in  − 80 °C.
9. The next day, transfer cryovials from  − 80 °C to liquid nitrogen storage.
Procedure for thawing cells
1. Preheat the medium in a 37 °C water bath.
2. Remove a vial of cells from liquid nitrogen and immediately place in a 37 °C water bath.
3. When the vial is almost fully defrosted, remove the vial and spray with ethanol.
4. Open the vial and add 500 µl of the medium dropwise.
5. Using a 5-ml pipette, gently aspirate the cells followed by 3 ml fresh medium.
6. Slowly add the medium with cells to a 15-ml Falcon tube containing 7 ml medium.
7. Use 1 ml of the medium to wash the cryovial and add it to the 15-ml Falcon tube.
8. Centrifuge at 200g for 5 min.
9. Aspirate away the medium and resuspend in 5 ml culture media and count the number of cells.
 CRITICAL STEP While defrosting keratinocytes for culture in EpiLife, it is advantageous to wash the pellet with EpiLife and spin
down a second time before seeding out to prevent excess differentiation induced by calcium or serum.
10. Adjust the concentration and seed out as required.

(iv) A  dd 1 ml of MEF-conditioned hES medium every following day. After 3–4 d, outgrowths of typical epithelial
keratinocytes are visible (Fig. 4b). At this point and anytime onward, it is possible to switch to EpiLife-based
medium if desired. If no outgrowth is observed after 5–6 d, it is unlikely to work.
(v) After 10–14 d, large colonies (up to 1 cm in diameter) are visible. At this stage, it is advisable to split the cells for
infection or subculture to avoid cells initiating contact-­dependent differentiation.
(vi) Infect and generate iPS cells as described in Steps 38–48 or, alternatively, as described in Box 4.
(B) Keratinocyte culture by trypsinization of plucked hair ● TIMING 10–14 d
(i) Coat 35-mm dishes or 12-well plates with Coating Matrix.
(ii) Collect appropriate amount of hair (typically 10 hair) in anagen growth phase (see Step 20) and rinse immediately in HBSS.
(iii) Using a 100-mm Petri dish, cut the ORS area into two to three pieces using a scalpel.
(iv) Add 2–3 ml TrypLE Select.
(v) Transfer into a 15-ml Falcon tube and incubate for 15 min, shaking very gently every 3 min.
 CRITICAL STEP After this stage, culture the cells as described in Box 1 if culture in serum/feeder-based approach
is needed or preferential.
(vi) Add 10 ml of EpiLife, pipette vigorously up and down for 10–15 times to obtain single cells detached from the
plucked hair.
(vii) Spin down cells for 5 min at 200g.
(viii) Count the number of cells (e.g., with a hemocytometer).
 CRITICAL STEP Cell yield will vary but typically 3–4 × 104 cells per hair can be expected. Too few cells may indicate
insufficient trypsinization. A second round of trypsinization may be required.
(ix) Seed out at a density of 2–3 × 103 cells per cm2 in EpiLife.
(x) Infect and generate iPS cells as described in Steps 38–48.

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 protocol

Box
  4 | DIRECT iPS CELL GENERATION BY INFECTION OF KERATINOCYTES
 AFTER OUTGROWTH FROM HAIR COLONIES ● TIMING 2–4 WEEKS
This strategy functions as a rapid and simple way to generate iPS cells from a single hair as shown17. If multiple iPS cell colonies
are required and the starting population needs to be preserved, performing EpiLife culture as described for hair followed by infection
as described in Step 22 followed by that in Steps 38–48 is recommended.
1. Obtain keratinocyte colonies that are grown from a hair in hES medium as described in Step 22(A).
2. Aspirate, wash with PBS and trypsinize the colony using 1 ml of 0.25% Trypsin/EDTA.
3. When cells have rounded up after approximately 5–8 min, gently tap the 35-mm dish to release all cells.
4. Resuspend in 10 ml hES medium.
5. Centrifuge at 200g for 5 min.
6. Resuspend the pellet in 4 ml MEF-conditioned hES medium.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

7. Seed out the cell suspension in two 35-mm dishes or one 60-mm dish that has been pre-coated with Matrigel.
8. The next day, add 2 ml of OSKM retrovirus (containing 10 µg ml − 1 polybrene).
9. Centrifuge dishes at 650g for 45 min.
10. Replace with 2 ml fresh MEF-conditioned hES medium (within 4–5 h).
11. The next day, repeat Steps 57–59 to infect the cells a second time.
12. Change the medium daily.
13. After 3–4 d, most non-infected keratinocytes will stop growing and start to differentiate into domes.
 CRITICAL STEP If seeded at too high a density and cells are growing fast, it may be required to trypsinize and reseed cells at this step.
14. After 1–2 weeks, large colonies that are visible can be picked mechanically and transferred onto irradiated MEFs or HFFs and
cultured as normal iPS cells according to the laboratory preference or as described in detail in Nature Protocols22.

Retrovirus production ● TIMING 1 week

23| Defrost a vial of Phoenix Ampho 293 cells. For details, see Box 3. Alternatively, if using other virus or production
approaches, switch to Step 38.
! CAUTION Use Category 2 (or higher) tissue culture hoods and exercise due caution in the production, storage and use of
recombinant retroviral particles.

24| Seed out ~2 × 106 cells in each 100-mm tissue culture dish.

25| On reaching 80–90% confluence (1–2 d), aspirate the medium, wash gently with PBS, again aspirate and add 2–3 ml of
0.05% Trypsin/EDTA. Incubate for 1 min at 37 °C and gently tap the tissue culture plate ensuring that all cells are in suspension.

26| Add 10 ml of complete DMEM medium and transfer to a 15-ml Falcon tube.

27| Centrifuge at 200g for 5 min. a

28| Resuspend in 10 ml of complete DMEM medium and
count the number of cells.

29| Seed out 4.3 × 106 cells in 10 ml DMEM medium in

100-mm culture dishes and place in a 37 °C, 5% CO2
incubator.

30| The next day, prepare the FuGENE/DNA complex

according to the manufacturer’s instructions. A ratio of
27 µl of FuGENE to 9 µg of plasmid DNA works well.
Alternatively, use the established transfection system of b
the laboratory.

Figure 4 | Isolation and culture of plucked human hair. (a) An example

of plucked hair in anagen (growth) phase. Note the presence of the hair
bulb and the outer root sheath (ORS, arrow). The presence of the ORS cells
around the hair shaft is essential for keratinocyte isolation and growth.
(b) An example of keratinocytes growing out of the ORS of a human hair. The
illustration is of a sample after 9 d of growth in Matrigel-coated plate and
feeder-conditioned hES medium. Note the strongly epithelial appearance and
tight clusterering of cells. Scale bars represent 500 µm (a), 250 µm (b).

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 protocol

Box
  5 | CULTURE OF KERATINOCYTES FROM SMALL BIOPSIES BY EXPLANT
  OUTGROWTH ● TIMING 1–3 WEEKS
When biopsies are small or cells are sensitive (e.g., our Fanconi anemia samples18), it may be preferential to grow keratinocytes
using feeders and explant outgrowth system. It should be noted that the epidermis can be separated from the dermis first,
as described in Steps 1–4, if both keratinocyte and fibroblasts need to be isolated. However, both cell types can be isolated
as described below by selective growth using RM +  medium for keratinocytes and complete DMEM medium for fibroblasts.
Procedure
1. Using sterile forceps and scalpels, cut the biopsy into 0.5-mm pieces.
2. Using tweezers, place ~8 to 10 pieces in each uncoated 60-mm tissue culture plate.
3. Add 1 drop of medium on each piece of tissue.
4. Gently transfer to a 37 °C, 5% CO2, 90% humidity incubator overnight (or minimum 5–6 h).
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

 CRITICAL STEP Do not allow pieces to dry out completely.

5. The next day, very gently add 5 ml of complete RM +  media containing 5 × 106 irradiated human fibroblasts. Mouse fibroblasts can
also be used and the mouse 3T3-J2 cell line (originally derived by Dr. Howard Green, Harvard Medical School, Boston, MA) gives the
best result.
 CRITICAL STEP If cells that are not contaminated by feeder cells are preferred, it is possible to use RM +  medium preconditioned by
feeders. Not adding feeder cells may reduce growth rate and yield, and increase the chance of contaminating fibroblasts growing out
from the pieces.
 CRITICAL STEP It is essential that the pieces of tissue remain attached to the plate. Floating pieces can be plated again in a new
dish following the above-mentioned steps.
? TROUBLESHOOTING
6. Carefully return the plate to the incubator.
7. Replace with fresh RM +  medium every 3–4 d and remove any pieces of tissue that are floating. Within 1 week, outgrowths of
keratinocytes should appear.
 CRITICAL STEP At this stage, and any passage after this, it is possible to switch to EpiLife medium as described in Step 20.
8. After 14–21 d, aspirate the media and wash two times with PBS.
9. Add 4–5 ml of 0.05% Trypsin/EDTA solution and incubate at 37 °C for 1 min to detach feeder cells.
10. Aspirate feeders and add 4–5 ml of 0.25% Trypsin/EDTA, incubate for 5–8 min at 37 °C until keratinocyte colonies have rounded up
and some of the cells are floating.
11. Tap the culture dish firmly on the side to release at least 95% of the cells.
12. Add 10 ml of fresh RM +  medium and transfer to a 15-ml Falcon tube.
13. Centrifuge at 200g for 5 min.
14. Passage at a ratio of 1:4 or 1:5 in plates that are pre-seeded with irradiated feeder cells.
15. Change the medium every 3 d and split the cells before reaching 80% confluence (typically once every week).
16. Use the cells for reprogramming experiments or freeze them as described in Box 3.

31| Add the FuGENE/DNA solution dropwise onto the medium (gently).

32| Place in a 37 °C, 5% CO2 incubator overnight.

33| The next day, gently change the medium (10 ml per plate) and incubate overnight at 32 °C in a 5% CO2 incubator.
 CRITICAL STEP Virus is more stable at 32 °C, resulting in higher infectivity, although 37 °C is acceptable.
 CRITICAL STEP At this time point, near 100% of cells should be transfected. Check using GFP reporter plasmid of
choice.

34| Collect the viral supernatant and add fresh complete DMEM medium to the dishes.
 CRITICAL STEP Take care to avoid cells detaching from the tissue culture plates.

35| Every following day, for 2–4 d, repeat Steps 33 and 34 in order to collect more viral supernatant.

36| Filter the viral supernatant through a 0.45-µm PVDF filter to remove any cells.
 CRITICAL STEP Use low-protein-binding filters to avoid trapping of virus and reduction of titer.
 PAUSE POINT Virus can be snap-frozen in cryovials using liquid nitrogen and stored at  − 80 °C or in liquid nitrogen for
several months. Some loss of infectivity is observed on freezing.

37| Add 1 µl of polybrene (10 mg ml − 1) for each milliliter of viral supernatant needed (giving a final polybrene concentration
of 10 µg ml − 1).

nature protocols | VOL.5 NO.2 | 2010 | 379

 protocol
Figure 5 | Infection of keratinocytes and derivation of iPS colonies. (a) An
example of keratinocytes 1 d after defrosting from cryovial and seeding
a b
50,000 cells per well of a six-well plate. Note the small clusters of three to
eight cells that allows efficient infection with retrovirus while preventing
excessive cell death. (b) GFP-positive keratinocytes 24 h after the second
round of infection. Most cells are infected and healthy. Note some flat and
differentiated nondividing cells that have not been infected. (c) An example
of primary iPS colonies (black arrow) 8 d after infecting keratinocytes with
OSKM and plating them on MEF feeder cells in hES medium. An example of a
non-reprogrammed keratinocytes is also visible (white arrow). (d) An example
of established KiPS colony. (e) The quality of KiPS colonies must be thoroughly
c d
examined using a variety of markers for pluripotency. The example here shows
the presence of alkaline phosphatase activity (in this case detected in blue).
(f) The capacity of KiPS cells to differentiate should also be tested. This
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

example shows the formation of embryoid bodies. Scale bars represent 250 µm.

Infecting keratinocytes and generating iPS colonies

● TIMING 2–4 weeks
38| Trypsinize or defrost keratinocytes at passages 0–5 and e f
seed out 50,000 cells per well in six-well dishes.
 CRITICAL STEP Keratinocytes cultured in EpiLife medium
are highly sensitive to prolonged exposure of serum or
calcium concentration above 0.06 mM. If exposed to serum
during freezing, wash the pellet with PBS, centrifuge again
and resuspend in EpiLife.

39| The following day, the cells should be ~30% confluent with small clusters of 4–8 keratinocytes (Fig. 5a). Add fresh
retroviral supernatant from Phoenix Ampho cells that have been filtered with polybrene added (10 µg ml − 1). Use 1 ml of each
transcription factor for each well in a six-well plate. Thus, when using the combination of OSKM, the total volume will be 4 ml.
 CRITICAL STEP The use of fresh supernatant is not essential but increases the efficiency of iPS generation.

40| Keep the cells for 10 min at 32 °C and then transfer the plate to a centrifuge and spin at 650g for 45 min at 32 °C.
 CRITICAL STEP Centrifugation of cells with viral supernatant significantly increases the infectivity under circumstances
where the retroviral supernatant must be removed after 1 h to prevent differentiation of cells. After centrifugation, leave
the cells for up to 20 min in an incubator.

41| Remove the supernatant and wash with PBS twice before replacing with fresh EpiLife.
 CRITICAL STEP Failure to remove the supernatant containing serum and high-calcium concentration will lead to a
significant number of keratinocytes senescing and differentiating.

42| After 24 h, repeat Steps 40 and 41 to infect the cells a second time. At 24 h after this stage, most cells apart from the
large, flattened differentiated cells, should be infected. This can be tested using GFP reporter-based retrovirus (Fig. 5b).
 CRITICAL STEP We have noted that some other, near-identical serum-free medium formulations available on the market,
which support the growth of keratinocytes equally well, result in significantly lower GFP intensity after infection.
? TROUBLESHOOTING

43| Plate out 4 × 106 irradiated MEFs in gelatin-treated 100-mm plates.

44| At 1–2 d after the second infection of keratinocytes, trypsinize the cells (in either TrypLE Express or Trypsin/EDTA) and
resuspend in 10 ml hES medium.

45| Centrifuge at 200g for 5 min.

46| Resuspend in hES medium and plate out infected keratinocytes in 100-mm culture plates treated with gelatin and
pre-seeded with 4 × 106 irradiated MEFs.

47| After 2 d, change the medium daily. After 4–5 d, small colonies are visible. Some colonies maintain typical keratinocyte
morphology and tend to differentiate, but colonies that are undergoing reprogramming are easily identifiable (Fig. 5c).

48| After 14–28 d, large reprogrammed colonies that grow fast are identifiable. These can be picked mechanically, subcloned,
cultured and characterized (Fig. 5d–f) according to laboratory preference or as described in detail in Nature Protocols22.
? TROUBLESHOOTING

380 | VOL.5 NO.2 | 2010 | nature protocols

 protocol

● TIMING
Steps 1–12, isolation of keratinocytes from foreskin samples or punch biopsies: 2 d
Steps 13–18, serum- and feeder-free culture of keratinocytes: 2–3 weeks (1–3 passages)
Steps 19–22, culture of keratinocytes from plucked hair: 2–3 weeks (1–2 passages)
Steps 23–37, retrovirus production: 1 week (two collections)
Steps 38–48, infecting keratinocytes in EpiLife and generating iPS colonies: 2–4 weeks (until mechanical picking
of iPS cells)

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

Table 1 | Troubleshooting table.

Step Problem Possible reason Solution

4 The epidermis sticks to the
dermis and does not easily
peel off as a single sheet
7 Few cells detach; solution
does not become turbid;
pieces of skin do not show
the signs of digestion
22(a) The hair does not stick to
the tissue culture dish
and floats in the medium
42 Poor retroviral transduction
efficiency
48 No iPS-like colonies appear
Box 2 The tissue does not stick
(Step 4) to the tissue culture dish
Box 5 and floats in the medium
(Step 5)
Tissue pieces too large
Insufficient enzymatic
digestion or old dispase
solution
Insufficient trypsinization
Tissue pieces too large
Culture conditions are
not optimized
Low viral titer or
unhealthy keratinocytes
Low infection-efficiency
or error in expression
constructs. Unhealthy
keratinocytes. Incorrect
culture procedure or
conditions
Culture conditions are
not optimized
Ensure that the pieces are not more than 5 mm in diameter. Place
tissues in fresh dispase solution and incubate at 37 °C for 1–3 h.
Test whether the epidermis can be peeled off every 30 min
Collect the solution and transfer tissue pieces to another tube
with fresh trypsin and incubate for another 20–30 min. Ensure
that pieces are  < 2 mm in size. Pipette vigorously after adding the
medium
It is very important to limit the amount of the medium during the
first 2–3 d so that the hair and the cells are properly in contact
with the dish yet do not dry out. On the first day, try to add only
a few drops of medium
Try to cut the ORS part of the hair into two to three smaller pieces
Add hair in a tissue culture dish pre-seeded with feeder cells,
which tend to promote rapid adhesion as well as keratinocyte
outgrowth
Physically pin down the hair either by (a) placing a glass cover-
slip above the hair or by (b) working on ice, dilute Matrigel with
cold hES medium to a concentration of 2 mg ml − 1. Pipette ~20 µl
of Matrigel directly on the follicle in the culture plate. Place the
plate in a 37 °C incubator for 15 min for the Matrigel to polymerize
before gently adding some more media
Viral titer is low: ensure that Phoenix cells are healthy and
transfection efficiency is close to 100%. Use low-passage Phoenix
cells. Passage Phoenix cells every few months in hygromycin
(300 µg ml − 1) and in Diphtheria toxin (1 µg ml − 1) for 1 week to
ensure the expression of packaging genes
Infectivity of other cell lines is high: adapt the keratinocyte culture
to achieve optimum healthy cell growth and ensure that cells are in
clusters of four to eight cells
Check that efficient infection of all transcription factors is
achieved. Try seeding keratinocytes on feeders in a range of 0–5 d
after the last viral transduction using different seeding densities.
Ensure that keratinocytes are healthy and at low passage. Perform
control infection of fibroblasts to ensure that viral constructs are
working
Limit the amount of medium on the first day so that the tissue
pieces are in proper contact with the dish yet do not dry out.
On the first day, try to add only a few drops of media
Try to cut the tissues in smaller pieces
Place a glass coverslip above the tissue until passage 1 to pin
down the tissue

nature protocols | VOL.5 NO.2 | 2010 | 381

 protocol

ANTICIPATED RESULTS
Keratinocyte isolation and culture
Keratinocytes will become highly proliferative around 1 week of seeding single cells. Keratinocytes have a highly typical
morphology and can be identified using a number of markers such as specific keratins for basal cells as well as a number of
specific keratins and cross-linking proteins and enzymes in differentiated cells. From a small biopsy, it is possible to obtain
several confluent 100-mm dishes within 3–4 weeks/1–2 passages. This is sufficient for multiple iPS cell generation experi-
ments at low passage. Younger samples tend to give better quality and a higher number of passages. From a foreskin sample,
the yield will be much higher, between 500,000 and 1 million cells, at isolation reaching 1–2 million within 7 d followed by
exponential doubling for every passage. From this, five to six passages (1:4) are easily achieved before significant changes
such as increased differentiation and senescence are observed. When seeding cryopreserved keratinocytes, expect ~90% of
cells to attach and initiate rapid proliferation within 24 h.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

Direct outgrowth of keratinocytes from plucked hair is typically seen within 3–4 d if successful. After 14 d, a colony of
cells (1 cm in diameter) from a single hair can be split and infected 2 d later in order to generate iPS cells. If larger amounts
of cells are required, subculture is possible as well as direct adaptation to EpiLife (at passage 0 or after). They typically
become confluent and are ready to split after 10–16 d. After this, they can be split 1:4 once per week with medium changes
every 2–3 d. When trypsinized, between 104 and 105 viable cells can be obtained from one hair at passage 0, which, depend-
ing on the culture technique, may be expandable up to 108 cells.

Keratinocyte infection and iPS generation

When infecting keratinocytes that are grown in EpiLife using the protocol described, around 90–95% of the cells are
expected to be infected after one to two rounds of infection. After infection and seeding onto MEF feeders, small iPS-like
colonies become visible very early on, typically within 4–6 d. During this period, several colonies of non-reprogrammed
keratinocytes are expected, which tend to differentiate and form layers of cells with domes in the center. Wait up to
3 weeks before picking the proper colonies to achieve high efficiency. KiPS generation is typically performed between
passages 0 and 3, although we have used cells up to passage 6. In rare cases, some KiPS colonies may differentiate more
easily than others or otherwise not behave as expected. In our experience, this is generally due to insufficient silencing
of the transgene, which can easily be checked with antibodies against FLAG if Flag-tagged transcription factor constructs
described in this protocol are used17.

Acknowledgments  We are grateful to Dr. Agustín Toll Abelló at the Hospital
del Mar (Barcelona, Spain) for facilitating collection of skin samples. We thank
Marta Berini Pérez for drawing Figure 1. This study was supported by grants
from Fundacion Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation,
Marato de TV3, CIBER and MICINN.
AUTHOR CONTRIBUTIONS  T.A. designed the protocol and all experimental
procedures, wrote the paper and prepared figures. J.C.I.B. is a project lead and
assisted with the preparation of the paper.
Published online at http://www.natureprotocols.com/. 13. Amoh, Y., Li, L., Katsuoka, K., Penman, S. & Hoffman, R.M. Multipotent
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
1. Fuchs, E. Scratching the surface of skin development. Nature 445,
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382 | VOL.5 NO.2 | 2010 | nature protocols