Cell Stem Cell
Generation of Functional Thymic Epithelium
from Human Embryonic Stem Cells
that Supports Host T Cell Development
Audrey V. Parent,1Holger A. Russ,1Imran S. Khan,1Taylor N. LaFlam,1Todd C. Metzger,1Mark S. Anderson,1,2,*
and Matthias Hebrok1,2,*
1Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143-0540, USA
2These authors contributed equally to the work
*Correspondence: email@example.com (M.S.A.), firstname.lastname@example.org (M.H.)
Inducing immune tolerance to prevent rejection is a
key step toward successful engraftment of stem-
cell-derived tissue in a clinical setting. Using human
pluripotent stem cells to generate thymic epithelial
cells (TECs) capable of supporting T cell develop-
ment represents a promising approach to reach
this goal; however,progresstowardgeneratingfunc-
tional TECs has been limited. Here, we describe a
robust in vitro method to direct differentiation of
human embryonic stem cells (hESCs) into thymic
epithelial progenitors (TEPs) by precise regulation
of TGFb, BMP4, RA, Wnt, Shh, and FGF signaling.
The hESC-derived TEPs further mature into func-
tional TECs that support T cell development upon
transplantation into thymus-deficient mice. Impor-
tantly, the engrafted TEPs produce T cells capable
of in vitro proliferation as well as in vivo immune
responses. Thus, hESC-derived TEP grafts may
have broad applications for enhancing engraftment
in cell-based therapies as well as restoring age-
and stress-related thymic decline.
The use of stem cells to replace lost or damaged tissue repre-
sents one of the most promising applications of stem cell
research. Among the most interesting and clinically relevant
cell types that still haven’t been successfully generated from
human pluripotent stem cells are thymic epithelial cells (TECs).
The thymus plays a crucial role in the immune system by sup-
porting the development of functional T cells. It is also the
main organ involved in establishing immune tolerance through
the elimination of autoreactive T cell subsets (reviewed in Ander-
son et al., 2007). Both of these critical functions are mediated by
TECs, the main component of the thymic stroma. Because the
thymus undergoes profound degeneration with age and when
exposed to stresses such as irradiation and chemotherapy, the
use of stem cells as a potential source of TECs to enhance or
restore thymic function is of great therapeutic interest. Given
the key role of TECs in establishing self-tolerance, differentiation
of a functional thymus from stem cells also has the potential
to enhance engraftment of human-stem-cell-derived tissue
through the induction of graft-specific immune tolerance. How-
ever, directed differentiation of human pluripotent stem cells
into TECs has not been successful to date and remains an
important challenge that needs to be addressed before such
approaches can be developed.
During embryogenesis, the thymus arises from the endoderm
of the third pharyngeal pouch, a specialized pocket of the ante-
rior foregut tube that contains the common primordium for the
prospective thymus and parathyroid glands (Le Douarin and
Jotereau, 1975; Gordon et al., 2004). The outgrowth of thymic
epithelium occurs from the ventral domain of the third pharyn-
geal pouch in response to developmental cues such as FGFs,
BMP4, and Wnt ligands (Balciunaite et al., 2002; Bleul and
Boehm, 2005; Patel et al., 2006). Crosstalk with lymphoid pro-
genitors that colonize the thymus subsequently allows differenti-
ation of common thymic epithelial progenitors (TEPs) into two
populations of mature TECs: cortical TECs (cTECs) and medul-
lary TECs (mTECs) (Rodewald, 2008).
Although previous studies have reported the successful differ-
entiation of human pluripotent stem cells into definitive endo-
derm (DE) and anterior foregut endoderm (AFE) (D’Amour
et al., 2005; Green et al., 2011), they failed to demonstrate sub-
sequent specification to the thymic lineage. Here we show that
in-vitro-directed differentiation of human embryonic stem cells
(hESCs) into TEPs can be achieved through recapitulation of
the embryonic signaling events that guide thymic development
in vivo. We have found that a precise temporal control of the
(Shh), and FGF signaling is required to efficiently generate TEPs
in vitro. Importantly, we demonstrate that TEPs derived using
this method mature into functional TECs that support T cell
development upon transplantation into athymic mice.
In-Vitro-Directed Differentiation of hESCs into TEPs
Even though the molecular mechanisms responsible for speci-
fying thymus fate are still uncertain, prior work has identified
the Foxn1 and Hoxa3 transcription factors as early and essential
regulators of thymus specification and differentiation of
TEPs into mature TECs (Manley and Capecchi, 1995; Nehls
et al., 1996). We therefore focused our efforts on developing a
Cell Stem Cell 13, 219–229, August 1, 2013 ª2013 Elsevier Inc. 219
stepwise protocol that recapitulates thymus organogenesis by
using FOXN1 and HOXA3 expression as readouts for thymic
As summarized in Figure 1A, hESCs were sequentially differ-
entiated into DE, AFE, ventral pharyngeal endoderm (VPE), and
TEPs. Wefirstuseda previouslydescribed methodto inducedif-
ferentiation into DE using activin A (D’Amour et al., 2005). At the
end of stage 1, the majority of the cells coexpressed SOX17 and
FOXA2, confirming efficient specification to DE (Figure S1A
available online). Next, to promote the development of anterior-
ized and ventralized endoderm competent to give rise to
FOXN1+HOXA3+TEPs, we added activators and inhibitors of
signaling pathways that have been shown to influence anterior-
posterior and ventral-dorsal identities of emerging definitive
endoderm (Zorn and Wells, 2009). We found that treatment of
hESCs with high levels of activin A for 5 days (stage 1), followed
by the addition of BMP4, RA, and the TGFb inhibitor LY364947
Figure 1. Directed Differentiation of hESCs
(A) Schematic of differentiation protocol and
marker genes for specific stages. ES, embryonic
stem cells; DE, definitive endoderm; AFE, anterior
endoderm; TEP, thymic epithelial progenitors;
TEC, thymic epithelial cells.
(B) Gene expression analysis of day 11 hESCs
treated with the indicated factor combinations
(conditions 1–7) (n = 4–10). Fetal and adult human
thymus samples served as controls. Values are
normalized to TBP, relative to undifferentiated
hESCs, and shown as mean ± SD. Dash lines
correspond to fetal expression levels that were
used as a guide to optimize the differentiation
protocol (*p < 0.05, **p < 0.01, ***p < 0.001,
unpaired Student’s t test, compared to undiffer-
(C) Immunofluorescence analysis of stage 4
cultures differentiated with condition 7 for HOXA3
(green) and EpCAM (red) protein expression.
Nuclei were stained with DAPI. Scale bar = 50 mm.
See also Figure S1.
(stage 2 and 3), and then BMP4 and RA
alone (stage 4), led to a significant in-
crease in FOXN1 and HOXA3 expression
over undifferentiated hESCs at the end of
stage 4 (Figure 1B, condition 6). In addi-
tion, hESCs differentiated under these
conditions expressed EYA1 and GCM2,
two markers found in the developing third
pharyngeal pouch (Figure S1B), thus con-
firming the formation of pharyngeal endo-
derm (PE) in our cultures. Interestingly,
HOXA3 and EYA1 expression levels
obtained with these culture conditions
were not as high as those observed with
other treatments (Figures 1B and S1B,
conditions 1–5). These observations sug-
gest that specification to the thymic line-
age occurs more efficiently when the
levels of expression of these key factors remain below a certain
threshold. Importantly, our results also reveal that the duration of
the activin A treatment, as well as the presence of BMP4 and RA
during stages 2–4, are crucial to induce high levels of FOXN1
expression (Figure 1B, conditions 2–6).
Next, to optimize the efficiency of differentiation of AFE to VPE
and TEPs, cells were differentiated up to stage 2 with condition 6
before being exposed to additional molecules involved in
pharyngeal pouch patterning or involved directly in the induction
of Foxn1 expression (Balciunaite et al., 2002; Frank et al., 2002;
Bleul and Boehm, 2005; Moore-Scott and Manley, 2005; Gordon
et al., 2010; Neves et al., 2012). We found that the simultaneous
addition of BMP4, RA, Wnt3a, FGF8b, and the Shh inhibitor
cyclopamine at stages 3 and 4 led to an even more robust induc-
tion of FOXN1, while maintaining levels of HOXA3 and EYA1
similar to those found in human fetal thymus (Figures 1B and
S1B, condition 7). Immunostaining and flow cytometry analysis
Cell Stem Cell
Generation of Functional TECs from hESCs
220 Cell Stem Cell 13, 219–229, August 1, 2013 ª2013 Elsevier Inc.
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Generation of Functional TECs from hESCs
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