1
Title: I
dentification of a distinct ductal subpopulation with self
-
renewal and differentiation
potential from the adult murine pancreas
Jacob R. Tremblay
1,2*
, Jose Ortiz
1,2*
, Janine C. Quijano
1
, Heather Zook
1,2
, Jeanne M. LeBon
1
,
Wendong Li
1
, Kevin Jou
1
, Walter Tsark
3
, Jeffrey R. Mann
3
, Mark Kozlowski
4
, David A. Tirrell
4
,
Farzad Esni
5
, Dannielle D. Engle
6
, Arthur D. Riggs
1
, Hsun Teresa Ku
1,2
*
*
1
Department of Translational Research and Cellular Therapeutics,
2
The Irell and Manella Graduate
School of Biological Sciences, Diabetes and Metabolism Research Institute and Beckman
Research Institute, and
3
Center for Comparative Medicine, City of Hope, Du
arte, California.
4
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California.
5
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
.
6
Salk Institute for Biological Studi
es, La Jolla, California
.
*These authors contributed equally.
*
*
To whom correspondence should be addressed: Department of Translational Research and
Cellular Therapeutics, City of Hope, Duarte, CA 91010. Tel: 626
-
218
-
1174;
E
-
mail
: hku@coh.org.
Author Contributions:
Study Design: J.R.T, J.O. and H.T.K. Data Collection: J.R.T., H.T.K.,
J.C.Q., J.O., H.Z., J.M.L., W.L. Key Reagents: W.T., M.K., J.R.M., D.A.T., F.E. A.D.R. Data
Analysis: J.R.T., H.T.K., J.C.Q., J.O., H.Z., W.L., K.J., W.T., A.D.R. Drafting Paper: J.R.T.
, J.O.,
H.T.K., J.C.Q., H.Z., D.D.E., A.D.R
.
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2
Competing Interest Statement:
The authors declare that they have no conflicts of interest with
the contents of this article. The content is solely the responsibility of the authors and does not
necessarily repre
sent the official views of the National Institutes of Health.
Classification
:
Biological Sciences
. Cell Biology.
Keywords
:
Pancreas, Ductal cells, Progenitor cells, Injury, Proliferation
This PDF file includes:
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Figures 1 to
8
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Abstract
Pancreatic ducts function to deliver digestive enzymes into the intestines. Upon injury, ducts can
become proliferative and contribute to tissue regeneration; however, the identity of the ductal cells
that contribute to these processes is unknown. We combi
ned fluorescence
-
activated cell sorting, a
methylcellulose
-
containing 3
-
dimensional culture, droplet RNA
-
sequencing, and a clonal lineage
tracing tool to identify and isolate a distinct subpopulation of pancreatic ductal cells that exhibit
progenitor cell
properties. These ductal cells are unique in that they form tightly
-
bound clusters
(termed FSC
mid
-
high
), with an average of 8 cells per cluster. FSC
mid
-
high
clusters comprise only about
0.1% of the total pancreas, are tri
-
potent for duct, acinar and endocr
ine lineages, and self
-
renew
robustly
in vitro
. Transcriptomic analysis of FSC
mid
-
high
clusters reveals enrichment for genes
involved in cell
-
cell interactions, organ development, and cancer pathways. FSC
mid
-
high
clusters
express embryonic pancreatic proge
nitor markers Sox9, Pdx1, and Nkx6
-
1 at both transcription
and protein levels. FSC
mid
-
high
clusters are resistant to enzymatic dissociation and survive severe
in
vivo
acinar injury, which induces formation of ductal rosettes that become proliferative withi
n 14
days. Thus, FSC
mid
-
high
clusters represent a small subset of ductal cells with progenitor cell
properties
.
These
rare
progenitor
-
like duct cell clusters have implications in pancreas regeneration
and tumor initiation/progression.
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Introduction
In the adult pancreatic epithelium, there are three major lineages of cells: acinar, duct, and
endocrine cells.
Acinar and ductal cells are responsible for secreting and transporting digestive
enzymes, respectively, to aid in nutrient digesti
on while endocrine cells secrete hormones to
regulate glucose homeostasis.
Adult pancreatic cells are mostly quiescent during steady state
1,2
.
However, when damage and stres
s occur to acinar and endocrine insulin
-
producing beta cells
3
-
6
,
which results in pancreatitis and diabetes, respectively, proliferation increases in not only acinar
and endocrine cells but also ductal cells
7,8
. Ductal cells have been implicated as a source of
progenitor cells that contribute to beta cell
neogenesis
9,10
or pancreatic adenocarcinoma
11
.
Although the
roles of adult ductal cells as progenitor cells are still controversial
12
-
15
, recent
emerging evidence has
strongly
suggested that
certain
subpopulation of
ductal cells
are
involved
in regeneration of endocrine cells in conditions of insulin resistance
16
and insulin
-
dependent
diabetes
17,18
.
However, the
fundamental
identifies
of these progenitor
-
like ductal cells
remain
unknown.
To identify
potentially
rare
adult ductal progenitor cells,
here
we employ
ed
an unbiased
fractionation strategy by using fluorescence
-
activated cell sorting (FACS)
on dissociated
murine
pancreatic cells
;
we
discover a tightly
-
bound
ductal
cell cluster (termed FSC
mid
-
high
cluster)
,
which
constitute
s
only
0.
1% of the total pancreati
c cells
,
as the fundamental
progenitor
-
like cells
in the
normal
adult murine pancreas.
The
FSC
mid
-
high
clusters
can self
-
renew and differentiate
in vitro
and
are resistant to
in vivo
acinar injury conditions
.
Our results highlight the highly
heterogeneous
nature of the adult ductal cells
,
which may explain the difficult
ies
in studying these
rare ductal
cells
in the past
.
Results
CD133
high
CD71
low
duct cells contain cell fractions with distinct morphologies.
Flow cytometry
can be used to distinguish individual cells or small clusters based on light scattering at different
angles: the detected light beam in line with the originating beam is defined
as forward scatter (FSC),
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and that in perpendicular as side scatter (SSC). FSC is indicative of particle size while SSC
complexity or granularity, and the combination of these two parameters has been useful to
distinguish various cell types
19
. We hypothesized that adult pancreatic progenitor cells may be
small in size with a high nuclear
-
to
-
cytoplasm ratio, similar to adult hematopoietic stem cells
20,21
,
owing to a generally quiescent state in homeostasis
22
. To test this hypothesis
, whole pancreata
from adult mice were dissociated and analyzed by flow cytometry.
We previously
established
that a ductal cell sub
-
population identified by
CD133
high
CD71
low
cell
-
surface
staining shows progenitor
-
like properties
in vitro
23
.
We therefore further analyzed t
he
CD133
high
CD71
low
duct
population based on FSC and SSC (Fig. 1a and Supplementary Fig. 1).
Four sub
-
populations emerged from the parent CD133
high
CD71
low
population: abbreviated hereafter
as FSC
low
, FSC
mid
-
low
, FSC
mid
-
high
, and FSC
high
. These 4 sub
-
populations constituted 1.27 ± 0.27,
0.47 ± 0.13, 0.12 ± 0.04 and 0.23 ± 0.16% of the total pancreatic population, and 53.5 ± 7.8, 28.2
± 2.4, 5.3 ± 0.5 and 7.5
± 5.8% of the gated CD133
high
CD71
low
population, respectively. Adding the
% total pancreatic cells from all 4 sub
-
populations amounted to 2.09%, which is within the range of
the parent CD133
high
CD71
low
population among total pancreatic cells reported in o
ur prior study
(average 2.4 ± 1.9%)
23
. To examine cel
l morphology, freshly
-
sorted sub
-
populations were
cytospun and stained with Wright
-
Giemsa to distinguish nuclei and cytoplasm. The FSC
mid
-
high
sub
-
population contained aggregates of cells with high nuclear
-
to
-
cytoplasm ratios that were highly
resistant to
enzymatic single cell dissociation (to be addressed later in the study), which we named
“small clusters” (Fig 1b; upper right panel). The FSC
low
sub
-
population was comprised of mostly
single cells with high nuclear
-
to
-
cytoplasm ratios, which we named “single” (Fig. 1b; upper left
panel). The “large” morphology in the FSC
high
population had two phenotypes: one was bi
-
nucleated
with pink cytoplasm (
Fig. 1b; lower right panel), suggestive of some acinar cells
24
expressing
CD133
high
on the cell surface, and the other was single
-
nucleated with purple cytoplasm. The
identity of the latter cell is currently unknown. The FSC
mid
-
low
population contained a mix
ture of the
aforementioned 3 cell morphologies, with mostly “elongated” morphology plus small
clusters
(Fig.
1b; lower left panel). These 4 morphologies were also identifiable under a phase
-
contrast light
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microscope (Fig. 1c). The diameters of these morpho
logies were measured; “single” had the
smallest diameters, while “elongated”, “small clusters”, and “large” had increasingly larger
diameters (Fig. 1d). Counting the 4 morphologies in each sorted sub
-
population revealed that
higher FSC was positively corre
lated with an increased proportion of morphologies with larger
diameters (Fig. 1e), confirming effective sorting based on FSC.
The FSC
mid
-
high
cell fraction is enriched for pancreas colony
-
forming units (PCFUs).
The 3D
colony assay developed in our labora
tory utilizes methylcellulose as a matrix
25
.
Methylcellulose is
a biologically inert material
26,27
that prevents reaggregation of the plated cells while allowing for
expansion into a colony. The advantage of using methylcell
ulose is that other basement membrane
extracts, such as Matrigel, or defined ECM proteins, such as laminin, can be added at lower
percentages below their gelling point
25
. We named a progenitor cell capable of giving rise to a
colony a “pancreatic colony
-
forming unit (PCFU)”, following benchmarks used by hematologists to
quantify hematopoietic stem cell activity according to their colony
-
forming efficiency
28
.
To quantify the PCFUs in each sub
-
population, freshly
-
sor
ted cells from each fraction were
plated into a colony assay (Fig. 2a). In the colony assay containing Matrigel (5% vol/vol) and
Rspondin
-
1 (RSPO1)
25
(herein Matrigel/RSPO1 colony assay), the FSC
mid
-
high
fraction had
the
highest PCFUs, as seen by the resulting colonies (total number of 3
-
week
-
old colonies, which we
called “Cystic”, among 500 plated units) (Fig. 2b). This result contrasted our original hypothesis
that the FSC
low
fraction was most consistent with a stem
cell morphology. The FSC
low
fraction
contained some PCFUs, but the colony
-
forming efficiency was approximately 7
-
fold lower than the
FSC
mid
-
high
population (Fig. 2b).
We previously showed that performing the colony assay using a hydrogel containing the
IK
VAV sequence from laminin (herein laminin colony assay) in the absence of Matrigel and RSPO1
led to increased endocrine and acinar lineage markers and decreased ductal lineage markers
25,29
.
These colonies were termed endocrine/acinar (E/A) colonies. In the laminin colony assay, the
FSC
mid
-
high
fraction also had the greatest number of PCFUs (Fig. 2c), with an approximately 250
-
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fold higher colony
-
forming efficiency than the FSC
low
fraction. Because the FSC
mid
-
high
fraction has
the highest colony forming efficiency, we focused our attention on the
FSC
mid
-
high
fraction for
subsequent analyses.
To determine lineage marker expression, we micro
-
manipulated and picked one
-
by
-
one
individual Cystic and E/A colonies and analyzed gene expression by microfluidic qRT
-
PCR
analysis. Consistent with our prior fi
ndings
25,29
, FSC
mid
-
high
derived colonies grown in the
Matrigel/RSPO1 relative to laminin colony assay expressed higher levels of ductal (
Prom1, Krt19,
Ca2
) and
endocrine progenitor (
Neurog3
) markers, and lower levels of acinar (
Cpa1, Cela1
) and
endocrine cell (
Ins2, Glut2
) markers (Fig. 2d). These results suggest that the FSC
mid
-
high
fraction
contains multipotent progenitors or lineage
-
restricted progenitor cells
. Protein expression of
CD133, C
-
peptide, Muc1, Krt19, E
-
cadherin, and Neurog3 was confirmed in the colonies (Fig. 2e
and Supplementary Fig. 2). Previous studies determined that co
-
expression of Sox9, Nkx6
-
1, Pdx1
in embryonic pancreatic multipotent progen
itor cells (MPCs) is necessary for pancreas
development
30,31
. We found cells in the colonies simultaneously expressing these three proteins
(Fig. 2e; lower right panel), mimicking pancreatic MPCs during organogenesis.
To determine which cell morphology was responsible for forming colonies, we micro
-
manipu
lated FSC populations into Matrigel/RSPO1 colony assay at 1 cell (or cluster) per well in
96
-
well plates (Fig. 2f). The small round and large granular cells did not form colonies, whereas
elongated cells had a 30% colony
-
forming efficiency and small cluste
rs a notable 72.3% efficiency.
Altogether, these data suggest that the multicellular clusters in the FSC
mid
-
high
fraction contain
functional progenitor cells.
PCFUs within the FSC
mid
-
high
fraction self
-
renew.
One of the defining properties of a progenitor
cell is its ability to self
-
renew. To test the self
-
renewal potential of the PCFU
-
enriched
FSC
mid
-
high
fraction
, a serial dissociation and replating strategy was used (Fig. 3a). In our previous studies, we
op
timized self
-
renewal conditions in our Matrigel/RSPO1 colony assay, in which exogenous
RSPO1 is necessary for PCFU self
-
renewal
25
. The FSC
mid
-
high
fraction was plated into the
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Matrigel/RSPO1 colony assay. The resulting 3
-
week
-
old primary colonies were dissociated and
serially replated for a total of 4 generations. In parallel experiments, we used the FSC
low
fraction as
a control due to its higher pr
oportion among parent cells (Fig. 1a) and lower PCFUs (Fig. 2b
-
c).
The FSC
mid
-
high
fraction grew exponentially from the first generation and gave rise to a higher
number of total cells (both PCFUs and non
-
PCFUs) across all generations compared to the FSC
lo
w
fraction (Fig. 3b
-
c). Although the FSC
low
fraction had a lag phase in the early passages, the growth
rate (slope of the curve) caught up with the FSC
mid
-
high
fraction in the later passages (Fig. 3b
-
c).
This “catch up” effect in later passages of the FSC
l
ow
population was in both the proportion of the
total cells (Fig. 3d) and total colonies (Fig. 3e) per well. Thus, even though the FSC
low
fraction had
fewer PCFUs to begin with, which could be elongated and/or small clusters, the PCFUs retain the
same self
-
renewal capacity as those in the FSC
mid
-
high
. These results are consistent with the FSC
mid
-
high
fraction being more enriched in PCFUs compared to the FSC
low
fraction (Fig. 2b
-
c). Overall,
after 9 weeks in the self
-
renewing culture condition, the total cel
l number and total PCFUs
contained in the FSC
mid
-
high
fraction expanded ~440,000 and ~78,000
fold, respectively (Fig. 3b
-
c).
This
fold expansion of PCFUs is comparable to the sorted CD133
+
Sox9
+
total ductal cells from our
earlier report
25
.
To determine whether PCFUs maintain multi
-
lineage potential after multiple passages
in
vitro
, differences in acinar, ductal, and endocrine lineage marker expression of individual colonies
between the 1
st
and 4
th
generations were compared by microfluidic qRT
-
PCR. There was no
observable difference in the morphology of cystic colonies (Fig. 3f) or gene expression (Fig. 3g)
between the 1
st
and 4
th
generation colonies. These results demonstrate that PCFUs in the sort
ed
FSC
mid
-
high
population retain tri
-
potency over multiple generations
in vitro
.
FSC
mid
-
high
clusters contain tightly
-
bound individual cells resistant to enzyme dissociation
in
vitro
.
The small clusters in the FSC
mid
-
high
fraction (referred to as FSC
mid
-
h
igh
clusters) are composed
of multiple cells, so we sought to dissociate them into single cells for subsequent studies. Because
we knew that FSC
mid
-
high
clusters were resistant to collagenase (which was used to dissociate the
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pancreas prior to sorting), we
employed trypsin. Remarkably, FSC
mid
-
high
clusters remained intact
even after a 1 hr incubation with 0.25% trypsin
-
EDTA (Fig. 4a), and retained their colony
-
forming
ability compared to untreated FSC
mid
-
high
clusters (Fig. 4b). Other enzymes, including Lib
erase and
TrypLE, were tested with similar results (data not shown). FSC
mid
-
high
clusters potentially resist
dissociation due to strong cell
-
adhesion properties
32
. To examine this, we performed
immunofluorescence staining for classical tight junction markers and found that E
-
cadherin, TJP1
(ZO
-
1), and F11r (JAM
-
A)
33,34
were expressed at cell
-
cell interfaces of the FSC
mid
-
high
clusters (Fig.
4c and Supplementary Fi
g. 3a). Interestingly, this was a feature found only in FSC
mid
-
high
clusters;
cystic colonies, formed after plating, expressed low levels of ZO
-
1 and JAM
-
A
(Supplementary
Fig.3b
-
c). Transmission electron microscopy (TEM) also revealed tight junctions at ce
ll
-
cell
boundaries of individual cells within FSC
mid
-
high
clusters (Fig. 4d, yellow arrows), and each cell within
the small cluster was single nucleated (Fig. 4d, nuclei labels). We counted the number of individual
cells with nucleus per cluster and found
that a cluster had an average of 8 cells (Fig. 4e and
Supplementary Fig. 4).
To visualize individual cells within the FSC
mid
-
high
clusters in 3
-
dimensional
(3D) space, we used serial block
-
face 3D scanning electron microscopy (3D
-
SEM). This confirmed
the f
inding that FSC
mid
-
high
clusters are comprised of multiple individual cells (Supplementary
Movie). Taken together, these results demonstrate that FSC
mid
-
high
clusters consist of individual
single
-
nucleated cells that are tightly bound.
Cell
-
tracing analys
is reveals that FSC
mid
-
high
clusters consist of cells with clonal potential
that form individual cystic colonies.
Despite the inability to dissociate FSC
mid
-
high
clusters, we
investigated whether colonies were derived from single or multiple cells. To test this, we employed
a random X inactivation system and generated
Hprt
DsRed/+
mice in which the gene for
Disocosoma
sp. red fluorescent protein (DsRed) replaced hy
poxanthine guanine phosphoribosyl transferase
(Hprt) on one X chromosome. In mammals, inactivation of one X chromosome takes place
randomly in the pre
-
implantation female embryo
35
and is somatically inherited with extreme fidelity
36
. As a result, hemizygous female
Hprt
DsRed/+
mice are cellular mosaics, and approximately hal
f of
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their cells should express DsRed. As expected, fluorescence analysis and flow cytometry of
pancreatic cells and splenocytes showed mosaic expression of DsRed in hemizygous female
Hprt
DsRed/+
mice (Supplementary Fig. 5). For our purposes, it is importa
nt to emphasize that X
inactivation is somatically heritable; the progeny of a DsRed
+
cell will always be red and the progeny
of a DsRed
-
cell will always be devoid of the fluorescent signal.
The FSC
mid
-
high
clusters from hemizygous female
Hprt
DsRed/+
mice were cytospun onto
slides, fixed, stained with DAPI, and analyzed (Fig. 5a). As expected, about half of the small clusters
from FSC
mid
-
high
fraction were mosaic (containing both DsRed
+
and DsRed
-
cells, 46.3%); in
contrast, a quarter of small clusters
were either fully labeled (DsRed
+
, 24.4%) or unlabeled (DsRed
-
, 29.3%; Fig. 5b
-
d). Given that mosaic cell clusters contained DsRed
+
and DsRed
-
cells, we
predicted that plating mosaic cell clusters could form one of the following two types of colonies.
Fir
st, mosaic colonies if multiple cells within a cluster proliferate to form the colony, showing non
-
clonality, or second, a fully labeled DsRed
+
or unlabeled DsRed
-
colony if an individual DsRed
+
or
DsRed
-
cell within a cell cluster can self
-
renew and proli
ferate clonally.
Small clusters were plated into our Matrigel/RSPO1 colony assay and cultured for 3 weeks
(Fig. 5a). Individual colonies were hand
-
picked, fixed, stained with DAPI, and examined under a
confocal microscope using optical slice
z
-
stacks (Fig. 5e). Total nuclei (DAPI
+
) and total DsRed
+
cells were quantitated in 3D
-
reconstructed colonies; image analysis software was used to
unbiasedly label DAPI
+
cells as DsRed
+
or DsRed
-
. We found that about half of the colonies were
either fully
labeled (43.8%, DsRed
+
) or unlabeled (46.9%, DsRed
-
), while only a few colonies were
mosaic (9.4%, Fig. 5e
-
g). These data indicate that the majority of the colonies are clonally derived
from a single cell within the originating small cluster. Interestingly
, the sizes of individual colonies
were not uniform (Fig. 5f), suggesting heterogeneous proliferative potential of the originating
progenitor cells. Taken together, these cell
-
tracing experiments demonstrate that a single cell within
a FSC
mid
-
high
cluster
possesses progenitor function, and we speculate that the neighboring cells may
play other functions, such as regulating the proliferative potential.
Droplet
-
based gene expression profiling reveals FSC
mid
-
high
clusters
express genes involved
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in cell
-
cell in
teractions, development, and cancer.
To investigate gene expression patterns of
the FSC
mid
-
high
clusters, we performed droplet
-
based RNA sequencing with barcoding
37
(referred
to as droplet RNA
-
seq) on both sorted FSC
mid
-
high
and FSC
low
control units. After running quality
control (Supplementary Fig. 6a
-
c), a total of 1,125 FSC
mid
-
high
and 598 sorted FSC
low
units were
analyzed (Fig. 6a). These two sets of data were merged, resulting in a total of 8 clusters (Fig. 6b
-
c
and Supplementary Fig. 6d
-
f). Consistent with our prior
finding that the parent CD133
high
CD71
low
population is comprised of ductal cells
23
, most of the clusters expressed ductal markers (
Sox9,
Krt23, Krt17, Spp1
), except cluster 7 which expressed immune cell genes (Fig. 6b and
Supplementary Fig. 6g
-
i). To validate the expression of lineage markers, we performed qR
T
-
PCR
on sorted FSC
low
and FSC
mid
-
high
fractions. As expected, both FSC
low
and FSC
mid
-
high
fractions
expressed higher levels of ductal (
Sox9
) and lower levels of acinar (
Amylase2a
) and endocrine
(
Insulin2
) markers,
compared to unsorted total pancreatic cells (Supplementary Fig. 7a
-
c).
Further analysis of the seven ductal clusters revealed that clusters 0 and 1 were most
enriched in the FSC
mid
-
high
fraction, while clusters 2, 3, 5, and 6 were most enriched in the FSC
low
fraction (Fig. 6d
-
e). Cluster
-
specific differentially
-
expressed genes (Supplementary Dataset 1) were
further analyzed by gene set enrichment analysis (GSEA) using Gene Ontology and KEGG
molecular signature databases (Supplementary Dataset 2 and 3). Com
mon among clusters 0 and
1, which were enriched in the FSC
mid
-
high
fraction, were up
-
regulated pathways involved in cell
-
cell
interactions/signaling and organ development (Fig. 6f
-
g and Supplementary Fig. 8a
-
b).
Interestingly, various cancer pathways were
significantly up
-
regulated in cluster 1, but not cluster
0 (Fig. 6f
-
g). In sharp contrast, most pathways that were up
-
regulated among clusters 2, 3, 5, and
6 were involved in metabolism, although pathways in cellular differentiation and proliferation were
occasionally noted in cluster 2 (Supplementary Fig. 8a
-
b). These results suggest functional
diversity and heterogeneous nature of ductal cells.
As previously mentioned, embryonic pancreatic MPCs are responsible for expansion and
differentiation into all
three main pancreatic lineages and they express the transcription factors
Sox9
,
Pdx1
, and
Nkx6
-
1
30,31
. We hypothesized that clusters 0 and 1 could be enriched with units
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https://doi.org/10.1101/2022.07.12.499653
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