Supplementary Methods
Cell culture and differentiation. H9 hESCs were cultured in feeder-free conditions on Matrigel (BD
Biosciences, San Jose, CA) in mTeSR1 medium (STEMCELL Technologies, Vancouver Canada). Cells
were passaged every 4-5 days using Versene (Life Technologies, Grand Island, NY). Two different
protocols were used to facilitate epithelial differentiation of hPSCs. Protocol 1 (Metallo et al. 2010):
Differentiation was initiated at a cell density of ~10,000 cells/cm2 where hPSCs cultured in mTeSR1 were
switched to unconditioned hESC medium (UM) supplemented with retinoic acid (UM+RA): DMEM/F12
containing 20% Knockout Serum Replacer, 1X MEM non-essential amino acids, 1 mM L-glutamine (all
from Life Technologies, Grand Island, NY), 0.1 mM -mercaptoethanol, and 1 μM all-trans retinoic acid
(Sigma-Aldrich, St. Louis, MO). Cells were cultured in UM+RA for 7 days, changing medium daily. RAtreated cells were detached using 2 mg/mL Dispase (Life Technologies, Grand Island, NY) and passaged
onto gelatin-coated plates in Keratinocyte Defined Serum-free Medium (K-DSFM, Life Technologies,
Grand Island, NY). Cells were cultured for 3 weeks in this medium. Protocol 2 (Lian et al. 2013): hPSCs
cultured in mTeSR1 were switched to unconditioned hESC medium (UM) supplemented with 6 μM Src
Family Kinase inhibitor, SU6656 (Sigma-Aldrich, St. Louis, MO) and cultured for 3 days. The resulting
K18+ simple epithelial cells were then enzymatically removed using Accutase (Life Technologies, Grand
Island, NY) and subcultured onto Matrigel (BD Biosciences, San Jose, CA) in K-DSFM supplemented
with 5% fetal bovine serum (Hyclone, Logan, UT). K18+ cells were further differentiated into K14+
keratinocyte progenitors after 4 days in UM supplemented with 1 μM all-trans retinoic acid and 10 ng/mL
BMP4 (Sigma-Aldrich, St. Louis, MO) followed by 10 days in K-DSFM supplemented with 5% fetal
bovine serum (Hyclone, Logan, UT). All cells were cultured in wells with a cross-sectional area of 9.5
cm2.
Flow cytometry. Cells were detached from culture plates using 0.25% Trypsin-EDTA, fixed in 1%
paraformaldehyde for 10 minutes at 37°C, and permeabilized in FACS buffer (PBS with 0.5% BSA, 0.1%
NaN3, and 0.1% Triton X-100). Samples were incubated overnight in FACS buffer and primary
antibodies; control samples were included using no primary antibody. After a 1 hour stain with a speciesspecific secondary antibody, cells were analyzed on a BD LSRII flow cytometer using BD FACSDiva
software. Primary antibodies used for flow cytometry were PE-conjugated mouse anti-Nanog (BD
Biosciences, San Jose, CA), rabbit anti-K14 polyclonal (Lab Vision, Fremont, CA), AlexaFluor 700conjugated mouse anti-K18 monoclonal (Abcor, Holland, MI), and AlexaFluor 405-conjugated anticaspase3 (Santa Cruz Biotechnology, Santa Cruz, CA). Species-specific secondary antibodies were
obtained from Life Technologies (Grand Island, NY) and were conjugated to AlexaFluor 488 dyes.
Absolute numbers of cells were determined using a hemacytometer.
Model fitting and sensitivity analyses. Sets of ODE kinetic models representing the dynamics of the
various cell types in culture were fit to cell subpopulation dynamics data using parameter estimation via
least squares regression in MATLAB 2011. Sensitivity analyses on were performed by varying each
parameter individually by 10% and the resulting change in K14+ cell yield as predicted by the model
References
Lian X, Selekman J, Bao X, Hsiao C, Zhu K, Palecek SP. 2013. A small molecule inhibitor of SRC
family kinases promotes simple epithelial differentiation of human pluripotent stem cells. PLoS
One 8(3):e60016.
Metallo C, Ji L, de Pablo J, Palecek S. 2010. Directed differentiation of human embryonic stem cells to
epidermal progenitors. Methods Mol Biol 585:83-92.