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A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation

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A balance between stem cell self-renewal and differentiation is required to maintain concurrent proliferation and cellular diversification in organoids; however, this has proven difficult in homogeneous cultures devoid of in vivo spatial niche gradients for adult stem cell-derived organoids. In this study, we leverage a combination of small molecule pathway modulators to enhance the stemness of organoid stem cells, thereby amplifying their differentiation potential and subsequently increasing cellular diversity within human intestinal organoids without the need for artificial spatial or temporal signaling gradients. Moreover, we demonstrate that this balance between self-renewal and differentiation can be effectively and reversibly shifted from secretory cell differentiation to the enterocyte lineage with enhanced proliferation using BET inhibitors, or unidirectional differentiation towards specific intestinal cell types by manipulating in vivo niche signals such as Wnt, Notch, and BMP. As a result, we establish an optimized human small intestinal organoid (hSIO) system characterized by high proliferative capacity and increased cell diversity under a single culture condition. This optimization facilitates the scalability and utility of the organoid system in high-throughput applications.
Optimized human intestinal organoid demonstrates enhanced stemness and increased cell diversity a Schematic of screening strategy to optimize cultures of human intestinal organoids. b Schematic of the targeting strategy to generate LGR5-mNeonGreen reporter system. c Medium composition comparison among ES, IF, IL, and TpC culture systems. ¹Noggin or BMP pathway inhibitor DMH1, ²R-Spondin1 conditioned media, ³WNT3a protein, WNT3a conditioned media or WNT surrogate. d Representative brightfield and fluorescence images of LGR5-mNeonGreen organoids cultured in IL patterning condition, IF condition or TpC condition. Representative images from three independent experiments. Scale bars, 200 μm. Quantification of LGR5-mNeonGreen proportion (e) and relative LGR5-mNeonGreen intensity (f) in IF, IL patterning and TpC organoids cultured for 4 weeks. n = 3 samples. Quantification of colony forming efficiency (g) and cell proliferation as indicated by the number of cells (h) in IF, IL patterning and TpC organoids cultured for ten days from single cells (8000 cells per well seeding). n = 4 samples. One-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. i Proportion of secretory lineage cells in IF and TpC organoids quantified by positive staining of LYZ, MUC2, and CHGA, respectively. Two-tailed unpaired t-test; data are presented as mean ± SD; n = 3 samples. j Representative images of enterocytes (ALPI), goblet cells (MUC2), enteroendocrine (CHGA), and Paneth cells (LYZ) in TpC organoids. Representative images from five independent experiments. k Representative confocal images of LGR5-mNeonGreen and DEFA5 positive Paneth cells in TpC organoid. White arrowheads indicate Paneth cells adjacent to LGR5 intestinal stem cells, red arrowhead points out a double-positive cell. Representative images from four independent samples. l The growth and mNeonGreen expression of a single LGR5-mNeonGreen⁺ cell cultured in TpC condition over 13 days. Also showing Paneth (LYZ), goblet (MUC2), and enteroendocrine (CHGA) cells in the 13-day colonies detected by immunofluorescence staining. Representative images from three biological replicates. Scale bars, (j–l), 50 μm. Arrowheads indicate the emergence of mNeonGreen expression at the same location. Source data for this Figure are provided as a Source Data file.
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ScRNA-seq analysis reveals cellular diversity and cell fate dynamics in TpC-organoids a UMAP plot showing clustering of cells from scRNA-seq analysis of TpC organoids. Cluster labels indicate cell types. LGR5.high and LGR5.low indicate intestinal stem cells with different levels of LGR5 expression; TA1, transit-amplifying cell type 1; TA2, transit-amplifying cell type 2; Sec Pre, Secretory Precursor; Sec Pre2, Secretory Precursor Type 2; EC, enterocyte; EEC, enteroendocrine cell. b Proportion of each cell type from scRNA-seq sample in a. c Dot plot showing expression and percentage of cells expressing cell type-specific markers in clusters from scRNA-seq analysis. Dot size and color indicate normalized gene expression level. d UMAP plots showing expression of cell type-specific markers in TpC organoids. Color intensity indicates normalized gene expression level. e MA plot showing differentially expressed genes between Paneth cells and LGR5-high cells. f Heatmap showing predicted PROGENy pathway activity for each cell cluster. g UMAP plot showing subset analysis of secretory cells along with connected cell clusters. h Pie chart showing the proportion of each secretory cell type. Total percentages of all secretory cell types equal 100%. i Dot plot showing expression and percentage of cells expressing cell type-specific markers in clusters shown in g. Dot size and color indicate normalized gene expression level. j UMAP plots showing expression of cell type-specific markers in the secretory subset. Color intensity indicates normalized gene expression level. k UMAP plots highlighting EEC cells and depicting expression of EEC subtype-specific markers. Color intensity indicates normalized gene expression level. l PAGA trajectory analysis depicting cell connectivity. Circle size indicates cell number, and line thickness indicates connectivity strength between cell clusters. m RNA velocity analysis using Dynamo showing inferred transition direction between cell states. n Pie charts (top) showing proportions of indicated cell types among total cells, and bar charts (bottom) showing the composition of indicated cell types from scRNA-seq analysis of ES, IF, IL, TpC organoids, and crypt.
… 
The combination of TSA, pVc, and CP induces enhanced stemness and increased cellular diversity in organoids a Brightfield and fluorescence images of organoids cultured for 1 week in indicated conditions. Representative images from three independent experiments. b, c Quantification of the percentage of LGR5-mNeonGreen cells (b) and relative fluorescence intensity (c) in organoids cultured in indicated conditions, n = 4 samples. Colony forming efficiency (d) and the number of cells (e) of organoids cultured in indicated conditions, n = 4 samples. f RT-qPCR quantification of cell markers expression levels, n = 3 samples. g Representative confocal images of LYZ, MUC2, and CHGA in organoids cultured in indicated conditions. Representative images from three independent experiments. h Proportions of secretory lineages in organoids cultured in indicated conditions, n = 4 samples. b–f and h, one-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. i Schematic of experiments in (j–o). j Confocal images of EdU staining of organoids. k Quantification of the percentage of LGR5-mNeonGreen cells and relative fluorescence intensity in organoids cultured under the indicated conditions, n = 3 samples. l Brightfield and fluorescence images of organoids cultured in indicated conditions. m EdU and immunofluorescence staining of CHGA, MUC2, and LYZ in organoid cultures as indicated. EdU was added for 1 hour before staining. Representative images of (j, l, m) were from three independent experiments. n FACS gating strategy of LGR5-mNeonGreen cells. o Quantification of EdU, LYZ, MUC2 and CHGA positive cell percentage as in (m), n = 4 samples. k and o, two-tailed unpaired t-test; data are presented as mean ± SD. p Quantification of the percentage of LGR5-mNeonGreen cells in organoids cultured under the indicated conditions, n = 6 samples. q LGR5-mNeonGreen fluorescence images of organoids cultured in indicated conditions. Representative images from four independent experiments. r Proportions of LGR5-mNeonGreen cells in organoids cultured in conditions as in (q), n = 4 samples. (p and r), one-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. Scale bars, (a and l), 200 μm; (g, j, m and q), 50 μm. Source data are provided as a Source Data file.
… 
IBET-151 reversibly promotes proliferation and inhibits secretory differentiation a Schematic of the screening strategy for modulating early EC enrichment in organoids. b Schematic of organoid culture conditions for experiments in (c–f). c Representative brightfield and fluorescence images of organoids cultured in indicated conditions. Representative images from three independent experiments. Scale bars, 200 μm. d Proportion of LGR5-mNeonGreen cells in organoids shown in (c). n = 5 samples. Two-tailed unpaired t-test; data are presented as mean ± SD. e EdU and immunofluorescence staining of Paneth cells (LYZ and DEFA5), goblet cells (MUC2), and enteroendocrine cells (CHGA) in organoids cultured as in (b). Representative images from three independent experiments. Scale bars, 50 μm. f Quantification of positive staining as in (e). n = 3 samples. Two-tailed unpaired t-test; data are presented as mean ± SD. g UMAP plot showing clustering of cells from scRNA-seq analysis of TpCI (TpC+iBET) organoids. Cluster labels indicate distinct cell types. h UMAP plots displaying the expression levels of cell type-specific markers (LGR5, MUC2, DEFA5, FGFBP1, OLFM4, CHGA, DEFA6, FABP2) in TpCI organoids. Color intensity indicates normalized gene expression levels. i Dot plot illustrating the expression and percentage of cells expressing cell type-specific markers across the clusters identified in (g). Dot size represents the percentage of cells expressing the marker, while color intensity indicates normalized gene expression levels. j Bar charts depicting the composition of indicated cell types from scRNA-seq analysis, comparing TpCI organoids with TpC organoids. Proportions of cell types are shown as percentages. k Cell type responsiveness to iBET treatment quantified by Augur, comparing the TpCI dataset with the TpC dataset. Augur scores for each cell type are displayed, indicating the degree of responsiveness to iBET treatment. l UMAP plot demonstrating pseudotime analysis and cell type labeling of MetaCells in the iBET dataset. Pseudotime was calculated using the Stavia package, and MetaCells were identified using the SEACells package. m Heatmap of cell type-specific marker gene expression along the pseudotime trajectory as shown in (l). The color bar indicates cell type labels consistent with (l). Source data are provided as a Source Data file.
… 
Directed differentiation of intestinal organoids via niche signaling modulation a Schematic of small molecules screening strategy for induction of differentiated cell types. Differentiation protocols for Paneth cells (b), goblet cells (c), enteroendocrine cells (d), and enterocytes (e) (upper panels) and representative confocal images of organoids after differentiation showing Paneth cells (LYZ), goblet cells (MUC2), enteroendocrine cells (CHGA) and enterocytes (ALPI) (lower panels). Representative images from three independent experiments. f–i Quantification of the percentage of differentiated cells as shown in (b–e) respectively. One-way ANOVA with Tukey’s multiple comparisons test; data are presented as mean ± SD; representative experiment showing n = 3 samples from each condition. j Schematic of differentiation conditions for organoids cultured in TpC and TpCI (iBET-treated) conditions for experiments (k and l). k Representative confocal images of differentiated organoids, showing Paneth cell (LYZ), goblet cell (MUC2), and enteroendocrine cells (CHGA) in TpC and TpCI organoids. Representative images from three independent experiments. l Quantification of the percentage of differentiated cells as in (k). Two-tailed unpaired t-test; data are presented as mean ± SD; representative experiment showing n = 3 samples from each condition. m Schematic and representative confocal images illustrating M cell differentiation in organoids cultured under TpC condition. Representative images from three independent experiments. Scale bars, 50 μm. Source data for this Figure are provided as a Source Data file.
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Article https://doi.org/10.1038/s41467-024-55567-2
A tunable human intestinal organoid system
achieves controlled balance between self-
renewal and differentiation
Li Yang
1,2,3,5
,XuleiWang
1,5
, Xingyu Zhou
1
, Hongyu Chen
1
, Sentao Song
4
,
Liling Deng
1
,YaoYao
1
&XiaoleiYin
1,2
A balance between stem cell self-renewal and differentiation is required to
maintain concurrent proliferation and cellular diversication in organoids;
however, this has proven difcult in homogeneous cultures devoid of in vivo
spatial niche gradients for adult stem cell-derived organoids. In this study, we
leverage a combination of small molecule pathway modulators to enhance the
stemness of organoid stem cells, thereby amplifying their differentiation
potential and subsequently increasing cellular diversity within human intest-
inal organoids without the need for articial spatial or temporal signaling
gradients. Moreover, we demonstrate that this balance between self-renewal
and differentiation can be effectively and reversibly shifted from secretory cell
differentiation to the enterocyte lineage with enhanced proliferation using
BET inhibitors, or unidirectional differentiation towards specic intestinal cell
types by manipulating in vivo niche signalssuchasWnt,Notch,andBMP.Asa
result, we establish an optimized human small intestinal organoid (hSIO) sys-
tem characterized by high proliferative capacity and increased cell diversity
under a single culture condition. This optimization facilitates the scalability
and utility of the organoid system in high-throughput applications.
Adult stem cell (ASC)-derived organoids are generated by mimicking
the intricate processes of tissue development, homeostasis, and
regeneration in vitro. They demonstrate a remarkable ability to reca-
pitulate aspects of the tissue structure, cellular composition, and
function, making them an attractive platform for studying develop-
ment and disease in vitro1,2. Despite signicant efforts, previous
attempts to culture ASC-derived organoids have encountered sig-
nicant challenges in replicating the complex and dynamic processes
that occur in vivo. Conventional organoid culture systems for many
tissues are optimized to maintain stem cell self-renewal for expansion,
resulting in decreased cellular diversity as cells remain undiffer-
entiated. Conversely, attempts to promote differentiation and
maturation often lead to cellular heterogeneity but limited pro-
liferative capacity, as seen in the liver, pancreas, lung, and other
tissues38. Thus, separate expansion and differentiation steps are
required for typical organoid cultures, which impedes their scalability
and utility in high-throughput screening.
Mouse intestinal organoid, the rst ASC-derived organoid system,
demonstrates remarkable parallel self-renewal and multidirectional
differentiation processes under the ENR condition9.However,achiev-
ing an equal balance in human intestinal organoids has been challen-
ging. Despite efforts to improve culture conditions to induce cellular
diversity while maintaining proliferation ability, achieving this balance
remains elusive. For example, Paneth cells, an essential cell type in the
Received: 4 August 2023
Accepted: 17 December 2024
Check for updates
1
Institute for Regenerative Medicine, State Key Laboratory of Cardiology and Medical Innovation Center, Shanghai East Hospital, Frontier Science Center for
Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
2
Institute of Biophysics, Chinese Academy of
Sciences, Beijing 100101, China.
3
College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
4
Department of Gastro-
enterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
5
These authors contributed equally: Li Yang, Xulei Wang.
e-mail: xyin@tongji.edu.cn
Nature Communications | (2025) 16:315 1
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
intestinal epithelium, are absent or rare in the improved IF culture
condition10 and require specic signals for their generation, such as
IL22, which alsoresults in decreased cell proliferation in the organoid11.
Moreover, the signals that regulate the self-renewal and multi-
directional differentiation of human intestinal stem cells (ISCs) have
yet to be determined.
Intestinal epithelial cells exh ibit remarkable plasticity, allowing for
continuous self-renewal, differentiation, and dedifferentiation along
the crypt-villi axis. For instance, ISCs maintain self-renewal by com-
peting for niche signals at the base of crypts and starting to differ-
entiate outside the crypt1214. At the same time, multiple differentiated
cell types could also revert to a stem cell state when re-entering the
stem cell niche1518. These processes occur in a highly dynamic manner
in vivo and are tightly regulated by intrinsic processes and extrinsic
niche signals.
Generating diverse and rapidly proliferating cells necessitates
stem cells with the capacity to generate multiple cell types and
orchestrate localized signaling gradients for spatially regulated self-
renewal and differentiation19,20. Achieving this remains challenging in
homogeneous organoid cultures lacking the spatial niche gradients
found in vivo. In this study, we hypothesize that enhancing organoid
stem cell stemness can amplify their differentiation potential, which
would increase the cellular diversity in organoids without applying
articial spatiotemporal signaling gradients. Furthermore, recreating
the dynamic modulation of cell fate observed in vivo in organoid
systems by regulating niche-intrinsic and cell-intrinsic signals may
facilitate this outcome. Here, we demonstrate that a combination of
small molecule pathway modulators can facilitate a controlled shift in
the equilibrium of cell fate towards a specicdirection,leadingto
controlled self-renewal and differentiation of cells. Additionally, we
have developed an optimized culture condition that captures the
delicate balance of self-renewal and differentiation of cells, resulting in
a highly proliferative organoid system with increased cellular diversity.
Results
Establishment of organoid system with enhanced stemness
To enhance cellular diversity in human intestinal organoids, our initial
goal was to increase the proportion of LGR5+stem cells rather than
driving differentiation directly. To achieve this, we sought to replicate
an in vitro niche for stem cells using a combination of small molecules
and growth factors (Fig. 1a). We generated an LGR5-mNeonGreen
reporter system to visualize LGR5+stem cells using CRISPR-Cas9
technology (Fig. 1b and Supplementary Fig. 1ac). We compared the
components used in previously identied culture conditions (Fig. 1c).
The ES condition facilitated the expansion of progenitor cells but
inhibited their differentiation, resulting in organoids devoid of secre-
tory cell types21. Although the improved IF condition enabled multi-
differentiation and self-renewal of the organoids, it was still associated
with limited cellular diversity, as evidenced by the absence or rarity of
mature enterocytes and Paneth cells10. The recently developed opti-
mized condition employs IL-22 to induce the generation of Paneth cells
based on the ES condition, but this comes at the cost of inhibited
organoid growth11.
Using the LGR5-mNeonGreen reporter, we nd that stem cells
under the IF and IL patterning conditions11 exhibit minimal expression
of LGR5-mNeonGreen (Fig. 1d) while IF condition capable of generat-
ing multiple secretory cell types (Supplementary Fig. 2b), which is
consistent with previous research. To promote the maintenance of
LGR5+stem cells while preserving their differentiation process, we
incorporated key factors utilized in mouse intestinal culture, including
EGF, the BMP inhibitor Noggin (or small molecule DMH1), and
R-Spondin1. We eliminated factors such as SB202190, Nicotinamide,
and PGE2, which have been demonstrated to impede the generation of
secretory cell types21. Additionally, we combined previously identied
niche factors, such as IGF-1 and FGF-210,andemployedCHIR99021asa
replacement for Wnt proteins, as it promotes theself-renewalof ISCs22.
We also included the ALK inhibitor A83-01, which has been shown to
promote cell growth21. This basal condition was utilized during the
screening process (Fig. 1a, c). A combination of 3 small molecules
(TpC), including Trichostatin A (TSA or T, an HDAC inhibitor), 2-
phospho-L-ascorbic acid (pVc or p, Vitamin C), and CP673451 (CP or C,
a PDGFR inhibitor) was found to substantially increase the proportion
of LGR5-mNeonGreen positive cells and their relative mNeonGreen
expression in the culture (Fig. 1d-f). Moreover, the colony-forming
efciency of dissociated single cells signicantly improved, and the
total cell count in the culture also considerably increased (Fig. 1g, h and
Supplementary Fig. 2a).
Generation of diverse and plastic cell types under the TpC
condition
Under the TpC condition, organoids could be efciently generated
from dissociated single cells, wherein scattered LGR5-mNeonGreen
expression was observed in each colony (Supplementary Fig. 2c).
Prolonged culture ledto extensive crypt-like budding structureswithin
the organoids, with budding structures containing Paneth-like cells
with dark granules (Supplementary Fig. 2d), indicating cell
differentiation.
Interestingly, under the TpC condition, multiple intestinal lineage
cells were readily generated, as evidenced by positive staining of
markers including mature enterocytes (intestinal alkaline phospha-
tase, ALPI), goblet cells (mucin 2, MUC2), enteroendocrine cells
(chromogranin A, CHGA), and Paneth cells (defensin alpha 5, DEFA5,
and lysozyme, LYZ) (Fig. 1i-k). Notably, we observed widespread
expression of LYZ mRNA in human intestinal organoids; however, at
the protein level, lysozyme specically marked Paneth cells, which co-
expressed DEFA5, another Paneth cell marker (Supplementary
Fig. 3ac).
Withinthe organoids, interspersed DEFA5-/LGR5+cells and DEFA5+
Paneth cells were observed, closely resembling their in vivo counter-
parts. DEFA5+/LGR5+cells were also observed co-existing in the orga-
noids, likely reecting the retention of LGR5-mNeonGreen expression
as LGR5+stem cells differentiated into Paneth cells (Fig. 1k). The bud-
ding area also exhibited extensive OLFM4 expression, a specicstem
cell marker for mouse intestinal stem cells23 (Supplementary Fig. 2e).
Moreover, the organoids displayed EEC subtype markers, including
somatostatin (SST) and glucagon (GCG) (Supplementary Fig. 2e).
Notably, LGR5+stem cells and secretory progenies, such as Paneth
cells, goblet cells, and enteroendocrine cells, were uniformly dis-
tributed in the organoids cultured under the TpC condition, both in
short-term (7-10 days) and prolonged cultures (3-4 weeks) (Supple-
mentary Fig. 2c, 2g, h and Supplementary Fig. 3d, e). The majority of
TpC organoids were budding-structured, although a small number of
round-shaped organoids also existed (Supplementary Fig. 3d, e). This
indicates a high degree of homogeneity between TpC-generated
organoids in terms of composition and structure, which is an advan-
tage for downstream applications. Additionally, the TpC condition
supported the generation and long-term maintenance of hSIOs from
multiple donors (Supplementary Fig. 2f), indicating the robustness of
this culture system.
To investigate cell growth dynamics, we longitudinally tracked
individual LGR5+stem cells under the TpC condition. Remarkably, we
observed that a single LGR5+stem cell could give rise to organoids
comprising various secretory cell types, such as Paneth cells, goblet
cells, and enteroendocrine cells (Fig. 1l). Intriguingly, we also detected
the loss and re-emergence of LGR5-mNeonGreen expression in the
organoids, indicative of dynamic differentiation and dedifferentiation
processes (Fig. 1l).
Intestinal cells possess remarkable plasticity16,18,24.Totestthe
ability of different cell types to initiate organoids, we sorted LGR5-
negative, low, and high cells and cultured them under the TpC
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 2
Content courtesy of Springer Nature, terms of use apply. Rights reserved
condition (Supplementary Fig. 4a, b). We found that LGR5-negative
cells had the highest colony forming efciency, with LGR5-positive
cells re-emerging in these organoids (Supplementary Fig. 4c, d). We
hypothesize that non-LGR5 cells, such as differentiating progenitor
cells and alternative stem cells like isthmus progenitor cells, could
serve as initiation cells for organoids25.Thesendings suggest that the
TpC condition supports generating a dynamic and plastic population
of intestinal cells in organoids, with the potential to give rise to mul-
tiple lineages and undergo cellular plasticity.
scRNA-seq analysis revealed cellular diversity and cell fate
dynamics in TpC-organoids
To gain insight into the cellular diversity of TpC-cultured organoids,
we conducted single-cell RNA sequencing (scRNA-seq) on the cells. We
DEFA5 LGR5 DAPI
LGR5
DEFA5
a
ih
TSA
pVc
CP
TSA
pVc
CP
Basal condition TpC TpC
Long-term
expansion
Combination
screening
EDRGA.IF.Ch
LGR5-
mNeonGreen
High LGR5+ propotion
High cellular diversity
Rapid proliferation
Highly efficient
expansion
Goblet cells
ISCs
Paneth cells
Enteroendocrine
cells
Enterocytes
Tuft cells
TA/Early ECs
b
cef
jd
l
LGR5-P2A-mNeonGreen
LGR5 promoter
Exon 18
LGR5
mNeonGreen
P2A
LGR5 promoter
Exon 18
LGR5
mNeonGreen
P2A
CpTLIFISE
EGF + + +/-
+/-
+++ +
IGF-1 -- +---+
FGF-2 -- +---+
Noggin1++ ++++ +
R-Spondin2++ ++++ +
A83-01 ++ ++++ +
WNT3high - high high high low -
SB202190 +- - +-- -
Nicotinamide +- -+-- -
Gastrin I ++--+
CHIR99021 -- --+-+
IL-22 ------
TSA -- ----+
pVc -- ----+
CP673451 -- ----+
Expansion Differentiation Expansion
(ES) Patterning Maturation Expansion &
Differentiation
Expansion &
Differentiation
+
ALPI
ALPI
Enterocyte
MUC2
MUC2 CHGA DAPI
CHGA
DAPI
Goblet EEC
LYZ
LYZ DAPI
Paneth
+
LGR5-mNeonGreen Brightfield
TpC
IL IF
g
k
LGR5-mNeonGreen
Day 0 Day 2 Day 4 Day 6
Day 8 Day 10 Day 11
LYZ DAPI
MUC2 CHGA DAPI
Day 13
IF IL TpC
0
2
4
6
8
LGR5-mNeonGreen (%)
P = 0.0010
P = 0.0008
IF IL TpC
0
500
1000
1500
Relative mean intensity
P = 0.0023
P = 0.0014
P = 0.2899
IF IL TpC
0
10
20
30
Colony forming efficiency (%)
P < 0.0001
IF IL TpC
0
2
4
6
Number of cells (×10
5
)
P < 0.0001
P < 0.0001
Paneth Goblet EEC
0
2
4
6
8
Percentage of cells (%)
IF
TpC
P < 0.0001
P < 0.0001
P < 0.0001
DAPI
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved
annotated the cells based on the expression of well-established mar-
kers and identied 12 distinct clusters of cells (Fig. 2a, b). Among these
clusters, we found that ISCs could be further divided into two sub-
populations based on their expression levels of LGR5 and OLFM4
(Fig. 2ad). Specically, we identied LGR5-high and LGR5-low ISCs,
two populations of cycling transit amplifying (TA) cells expressing low
levels of LGR5 but high levels of cell cycle genes such as MKI67 and
PCNA, a cluster of FABP1+early enterocytes (early EC) lacking LGR5
expression, mature enterocytes (EC) expressing ALPI and ACE2,and
multiple secretory cell types, including Paneth cells (DEFA5,DEFA6),
goblet cells (MUC2,SPINK4), enteroendocrine cells (CHGA,NEUROD1),
and tuft cells (ALOX5,AVIL)(Fig.2c, d). Of particular interest, we
identied two distinct secretory precursors. The rst (Sec Pre)
expressed classical secretory precursor markers such as HES6,DLL1,
and DLL4, representing typical secretory precursors. The second (Sec
Pre2) expressed OLFM4 as well as markers of both secretory (goblet,
Paneth, and enteroendocrine cell markers) and enterocyte lineages
(FABP1 and KRT20)(Fig.2c). We inferred that these cells represent
transdifferentiated cells from enterocyte lineages.
Differential expression analysis conrmed the upregulation of
classical markers for each cell type relative to LGR5-high ISCs (Fig. 2e
and Supplementary Fig. 5a). Notably, DEFA5,DEFA6,andPRSS2
exhibited the most marked fold change for Paneth cells. PROGENy
pathway activity analysis revealed distinct pathway activities for each
cell type. LGR5+stem cellsexhibited high Wnt activity, whereas TA cells
showed high MAPK pathway activity (Fig. 2f). To better understand
secretory lineage cells, we performed a subset analysis of secretory
cells (Fig. 2gk). Goblet cells were the most abundant, accounting for
32.3% ofall secretory lineage cells,consistent with their composition in
vivo. Paneth cells were less abundant, accounting for 8.6% of the
secretory lineage cells, while a small number of tuft cells were also
detected (1.4%) (Fig. 2h). These cells expressed classical markers
(Fig. 2ik). Enteroendocrine cells accounted for 24.7% of all secretory
lineage cells and expressed multiple subtype markers, including
NEUROG3 (progenitors). Among these subtypes, TPH1+enter-
ochromafnandCCK+I cells were the most abundant, consistent with
their in vivo abundance26,27. We also observed similar expression pat-
terns as previously reported, where GHRL+cells co-expressed MLN10,28
and mutually exclusive expression for MLN and GAST,forGHRL and
CHGA,andforTPH1 and GCG27. This suggests a high similarity of our
organoid with the in vivo intestine.
Notably, the Uniform Manifold Approximation and Projection
(UMAP) plot of TpC organoid-derived cells suggests a differentiation
path of secretory cells from LGR5-high ISCs to secretory precursors
(Sec Pre), partially differentiated LGR5-low/early ECs to the second
population of secretory precursors (Sec Pre2), as well as more differ-
entiated early ECs/mature ECs to goblet cells, in both the whole
organoid dataset (Fig. 2a) and the secretory subset (Fig. 2g). These
connections were also captured using the force atlas layout
(Supplementary Fig. 5b) and PAGA trajectory analysis (Fig. 2l), where a
major source of secretory lineage cells is a path from LGR5-high ISCs
towards secretory precursors (Sec Pre), which was conrmed by RNA
velocity analysis using the Dynamo package (Fig. 2m). This observation
is consistent with scRNA-seq data from freshly isolated intestinal
biopsies29, where Paneth cells and secretory lineages were found to
descend from LGR5+stem cells, and with ENR organoid data from
mouse organoids30. These trajectories are also present in fresh crypt
data, but not in IF10 or IL22 organoid datasets11 (Supplementary Fig. 5c),
where a possible source of secretory cells is partially differentiated
early ECs (Supplementary Fig. 5c). Notably, when we compared the
pathway activity of ISCs from different samples, we found that ISCs in
the TpC condition mostly resembled ISCs from freshly isolated crypt
samples (Supplementary Fig. 5e). To compare the cell composition of
organoids cultured under different conditions, we integrated scRNA-
seq datasets of cells cultured under multiple organoid conditions
using the Seurat package (Supplementary Fig. 5d). This analysis con-
rmed our previous observation that an LGR5-high population con-
nects absorptive and secretory lineages, which was present in the
Crypt data but mostly absent in the IF and IL22 samples (Supplemen-
tary Fig. 5d). Furthermore, the cell composition comparison conrmed
that TpC organoids capture major secretory cell types, including
Paneth, goblet, enteroendocrine, and tuft cells (Fig. 2n). We further
compared organoid data with in vivo data (Supplementary Fig. 6a).
Harmony analysis showed that most cell types were similar and co-
clustered with in vivo data. EC lineage cells, including early and mature
EC cells,formed distinct populations in all organoid conditions (ES, IF,
IL22, and TpC) and in vivo enterocytes. However, only TpC organoids
had signicant numbers of EC cells that co-clustered with the in vivo EC
cluster (Supplementary Fig. 6b, Supplementary Fig. 7, Supplementary
Fig.8).Webelievethismightbeduetospecic culture conditions,
such as high Wnt and low BMP levels, which inuence the properties of
EC cells inorganoid cultures. Additionally, cells in organoids exhibited
similar marker gene expression to those in vivo cell types, with TpC
organoids showing the highest similarity (Supplementary Fig. 9).
These results demonstrate the delity of our organoid system in
recapitulating the cellular diversity of the intestinal epithelium.
Small molecules play distinct roles in supporting stemness and
differentiation
Our TpC condition maintains cellular diversity and self-renewal of
LGR5+stem cells, providing a platform to dissect the roles of indi-
vidual chemicals in human intestinal cell fate regulation. The basal
molecules in the TpC condition are well characterized, including
EGF, IGF-1, FGF-2, the BMP inhibitor DMH1, R-Spondin 1, and A83-01,
which have been shown to play essential roles in maintaining mouse
and human intestinal organoids. Another molecule, CHIR99021, a
GSK3 inhibitor, activates the Wnt pathway and promotes ISC self-
renewal22.
Fig. 1 | Optimized human intestinal organoid demonstratesenhanced stemness
and increased cell diversity. a Schematic of screening strategy to optimize cul-
tures of human intestinal organoids. bSchematic of the targeting strategy to
generate LGR5-mNeonGreen reporter system. cMedium composition comparison
among ES,IF, IL, and TpC culturesystems. 1Nogginor BMP pathway inhibitor DMH1,
2R-Spondin1 conditioned media, 3WNT3a protein, WNT3a conditioned media or
WNT surrogate. dRepresentative brighteld and uorescence images of LGR5-
mNeonGreen organoids cultured in IL patterning condition, IF condition or TpC
condition. Representative imagesfrom three independent experiments. Scale bars,
200 μm. Quantication of LGR5-mNeonGreen proportion (e) and relative LGR5-
mNeonGreen intensity (f) in IF, IL patterning and TpC organoids cultured for
4 weeks. n= 3 samples. Quantication of colony forming efciency (g) and cell
proliferation as indicated by the number of cells (h) in IF, IL patterning and TpC
organoids cultured for ten days from single cells (8000 cells per well seeding).
n= 4 samples. One-way ANOVA with Dunnetts multiple comparisons test; data are
presented as mean ± SD. iProportion of secretory lineage cells in IF and TpC
organoids quantied by positive staining of LYZ, MUC2, and CHGA, respectively.
Two-tailed unpaired t-test; data are presented as mean ± SD; n=3 samples.
jRepresentative images of enterocytes (ALPI), goblet cells (MUC2), enteroendo-
crine (CHGA), and Paneth cells (LYZ)in TpC organoids.Representative imagesfrom
ve independent experiments. kRepresentative confocal images of LGR5-
mNeonGreen and DEFA5 positive Paneth cells in TpC organoid. White arrowheads
indicate Paneth cells adjacent to LGR5 intestinal stem cells, red arrowhead points
out a double-positive cell. Representative images from four independent samples.
lThe growth and mNeonGreen expression of a single LGR5-mNeonGreen+cell
culturedin TpC conditionover 13 days. Also showing Paneth (LYZ), goblet(MUC2),
and enteroendocrine (CHGA) cells in the 13-day colonies detected by immuno-
uorescence staining. Representative imagesfrom three biologicalreplicates. Scale
bars, (jl), 50 μm. Arrowheads indicate the emergence of mNeonGreen expression
at the same location.Source data for this Figure are provided as a Source Data le.
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 4
Content courtesy of Springer Nature, terms of use apply. Rights reserved
In our study, we focused on the roles of TSA, pVc, and CP. When
TSA, pVc, and CP were added individually or in combination to the
basal condition, TSA and CP signicantly increased the LGR5-
mNeonGreen percentages, with CP showing the most pronounced
effect on cell proliferation, colony formation from single cells, and
LGR5-mNeonGreen expression (Fig. 3ae). TSA and CP also boosted
the expression of Paneth cell markers (DEFA5,DEFA6,LYZ)andstem
cell markers (LGR5,SMOC2), with the TpC condition showing the
highest levels (Fig. 3f). CP also increased secretory cell diversity when
added to thebasal condition(Supplementary Fig. 10a), probably owing
to increased stem cells in the organoids.
We further investigated the effects of TpC factors by omitting
each factor from the TpC combination when initiating organoids.
Excluding any molecule from the TpC condition reduced ISC self-
PRSS2
DEFA6
TSPAN8
ITLN1
REG1A
PIGR
TFF3
DEFA5
REG3A
OLFM4
−3
0
3
6
9
024
Average expression
Log2fold change
Up: 31 Down: 13 NS
Paneth vs. LGR5-high
LGR5 OLFM4 ASCL2
ALPI
MCM6 MKI67
MUC2 CHGA DEFA5
ACE2
PROGENy pathway activity
LGR5.high
LGR5.low
TA1
TA2
Sec Pre
Goblet
Paneth
Tuft
EEC
Sec Pre2
Early EC
EC
WNT
TGFb
VEGF
p53
Tra i l
MAPK
EGFR
PI3K
NFkB
JAK−STAT
TNFa
Hypoxia
−1 01
ca
d
l
f
hg
j
k
nm
i
e
b
HES6
MUC2
CHGA NEUROD1
SPINK4DLL1
Sec Pre
Goblet
EEC
ALOX5 AVIL
Tuft
DEFA5 DEFA6
Paneth
PRSS2
NEUROD1CHGA
SSTGCG PYY
NPW
GHRL MLN
TPH1
CCK
NEUROG3
GAST
0
1
2
3
4
5
leiden
EEC cluster
Sec Pre Goblet Paneth EEC Tuft EC
HES6
DLL1
DLL4
ATOH1
MUC2
SPINK4
FCGBP
TFF3
CLCA1
DEFA5
DEFA6
PRSS2
REG1A
REG3A
CHGA
NEUROD1
CPE
RIMS2
SCG2
CCK
TPH1
ALOX5
AVIL
LRMP
POU2F3
FABP1
KRT20
Sec Pre
Goblet
Paneth
EEC
Tuft
Sec Pre2
Early EC
EC
20 40 60 80100
Percent expressed
01
Mean expression
20 40 60 80100
Percent expressed
01
Mean expression
LGR5
ASCL2
OLFM4
SMOC2
PCNA
HELLS
MCM6
MKI67
TOP2A
HES6
DLL1
DLL4
MUC2
SPINK4
DEFA5
DEFA6
ALOX5
AVIL
CHGA
NEUROD1
FABP1
KRT20
ACE2
LGR5.high
LGR5.low
TA1
TA2
Sec Pre
Goblet
Paneth
Tuft
EEC
Sec Pre2
Early EC
EC
ISC TA1 TA2 Sec Pre GC PC Tuft EEC EC
EC
Early EC LGR5.high
Sec Pre2
LGR5.low
TA1
EEC
TA2
Goblet
Paneth
Sec Pre
Tuf t
UMAP1
UMAP2
EC
EEC
Early EC
Goblet
LGR5.high
LGR5.low
Paneth
Sec Pre
Sec Pre2
TA1
TA2
Tuft
UMAP1
UMAP2
Goblet
32.3%
EEC
24.7%
Sec Pre2
18.3%
Sec Pre
14.7%Paneth
8.6%
Tuft
1.4%
EC
EEC
Early EC
Goblet
LGR5.high
LGR5.low
Paneth
Sec Pre
Sec Pre2
TA1
TA2
Tuft
TA2
6.2%
TA1
10.6%
EC
16.1%
Early EC
22.6%
LGR5.low
29.2%
LGR5.high
3.9%
0.2 Tuf t
1.0 Paneth
1.7 Sec Pre
2.1 Sec Pre2
2.8 EEC
3.7 Goblet
Total = 100.0%
Total = 100.0 %
(%)
11 8
20
112 13
15
2
24 10
37
4
32
2
100
74 67 73 76
48
0
25
50
75
100
ES IF Crypt IL22− IL22+ TpC
Proportion of cells (%)
Celltype
M/BEST4+
Paneth
EEC
Tuft
Goblet
0.6% 13.2% 15.5% 9.1% 12.6% 7.7%
Percentage of total cells
Fujii et al., 2018 He et al., 2022 This study
max
min
max
min
max
min
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved
renewal, as evidenced by the percentage of LGR5-mNeonGreen high
cells and their mean uorescence intensity (Supplementary
Fig. 10b, c). Although excluding TSA and pVc did not affect cell growth,
the absence of CP signicantly impacted organoid growth (Supple-
mentary Fig. 10b). Excluding TSA from the TpC combination markedly
decreased Paneth and EEC cells while increasing gobletcells. Excluding
pVc decreased Paneth cells but had little effect on EEC and goblet cells.
This could be attributed to the loss of ISCs capable of generating
Paneth cells and EECs (but not goblet cells) under conditions lacking
TSA or pVc. In contrast, excluding CP increased Paneth and EEC cells
but reduced cell proliferation, possibly due to immediate differentia-
tion of newly expanded ISCs in this condition (Fig. 3g-h). The combi-
nation of TpC demonstrated the best overall performance in organoid
growth, colony formation, LGR5-mNeonGreen expression, and secre-
tory cell diversity (Fig. 3ah, and Supplementary Fig. 10b, c).
We then evaluated the effects of withdrawingfactors from already
established TpC organoids. Withdrawing CP in this context led to
decreased cell proliferation (Fig. 3i, j, o), decreased LGR5 expression
and LGR5 cell proportion (Fig. 3k, l, n). It also increased cell differ-
entiation, conrmed by immunostaining for Paneth, goblet, and EEC
markers in the organoids (Fig. 3m, o), suggesting a role for CP in
preventing ISC differentiation, consistent with the effects of CP on
organoid generation from single intestinal cells.
TSA enhances LGR5 stem cell maintenance by targeting HDAC-
MBD-NuRD complex
To further dissect the mechanism of TSA, which is a pan-HDAC inhi-
bitor, we tested multiple HDAC inhibitors and found they similarly
promoted LGR5-mNeonGreen expression and increased cellular
diversity, albeit with varying degrees of efcacy (Supplementary
Fig. 11ad). Another pan-HDAC inhibitor, VPA, also increased the LGR5-
mNeonGreen proportion but signicantly decreased organoid growth
(Supplementary Fig. 11a). HDAC6 inhibitor Tubastatin A and HDAC2
inhibitor CAY10683 also signicantly increased the LGR5-mNeonGreen
proportion and intensity, similar to TSA (Supplementary Fig. 11ad),
while Tubastatin A did not affect Paneth cell generation (Supplemen-
tary Fig. 11e). These results suggest that TSA might promote stem cell
maintenance and Paneth cell differentiation through HDAC1/2. To
further investigate the target chromatin complex, we used small
molecules to regulate complexes containing the HDAC1/2 subunit. We
found that KCC-07 (KCC), an MBD2 inhibitor that prevents MBD2 from
binding to methylated DNA, could rescue the budding ratio and LGR5-
mNeonGreen expression in the absence of TSA (Fig. 3p and Supple-
mentary Fig. 12ad). Organoids treated with KCC-07 showed a greatly
increased frequency of Paneth cells compared to the -TSA condition
(Supplementary Fig. 12e, f). The expression of cell type-specic mar-
kers further conrmedthesimilareffectsofKCC-07andTSA(Sup-
plementary Fig. 12gk). MBD2 is a core subunit of the Nucleosome
Remodeling and Deacetylation (NuRD) complex, which includes
HDAC1/2 proteins. These results suggest that TSA targets the MBD2-
NuRD complex to promote LGR5-mNeonGreen expression and sub-
sequent Paneth cell differentiation.
The Methyl-CpG-binding domain (MBD) protein family includes
two members, MBD2 and MBD3. Previous studies have implied the
MBD3-NuRD repressor complex inhibits Lgr5 expression and pro-
genitor proliferation in the mouse intestine31. We inferred that MBD3-
NuRD also mediated TSA-induced gene expression in the human
intestine. To test this, we used CRISPR-Cas9 to genetically interfere
with MBD3 expression in human intestinal cells, using guide RNA tar-
geting exon 3 of human MBD3 due to the lack of a specic MBD3
inhibitor (Supplementary Fig. 13a). We isolated an independent clone
with reduced MBD3 expression, conrmed by Western blotting and
qPCR (Supplementary Fig. 13b, c). Knockdown of MBD3 signicantly
increased LGR5 expression (Supplementary Fig. 13c) and the propor-
tion of LGR5-mNeonGreen stem cells (Fig. 3q-r; Supplementary
Fig. 13d, e). Notably, LGR5 expression and the proportion of LGR5 stem
cells were signicantly higher in MBD3 knockdown organoids than in
WT organoids, even under TSA or KCC-07 conditions (Fig. 3q-r; Sup-
plementary Fig. 13d, e). Simultaneous inhibition of both MBD2 and
MBD3 (MBD3-KD + KCC-07) effectively enriched LGR5-mNeonGreen
stem cells. In summary, these experiments conrmed that TSA, as an
HDAC1/2 inhibitor in human intestinal organoids, led to MBD2 and
MBD3 NuRD-mediated enrichment of LGR5 stem cells, consequently
enhancing Paneth cell abundance.
We further performed single-cell RNA-seq to explore the effect of
TSA on different cell types. The withdrawal of TSA from established
TpC organoids (Supplementary Fig. 14a) induced premature differ-
entiation and subsequent death of Paneth cells in the organoids, as
indicated by the presence of a signicant number of Paneth cell marker
(DEFA5,DEFA6,andPRSS2) expressing cells with low UMI counts and
feature numbers (Supplementary Fig. 14b, c). These cells also co-
expressed markers of progenitors such as OLFM4,FABP2,andFGFBP1
(Supplementary Fig. 15a, b, e). Cell type priority analysis indicated that
Paneth cells, secretory precursor and LGR5-high cells were most sig-
nicantly affected by TSA withdrawal (Supplementary Fig. 15c, d).
Pseudotime and trajectory analysis indicated a similar cellular differ-
entiation path through LGR5 cells to secretory cells (Supplementary
Fig. 15f, g), where Metacell analysis revealed the topology of cellular
differentiation. LGR5 cells form a center that connects secretory cells
with TA cells and enterocyte lineages (Supplementary Fig. 15h-i), which
express typical cell type markers along pseudotime (Supplementary
Fig. 15j). These results are consistent with the role of TSA in LGR5 cell
maintenance and preventing the premature differentiation of ISCs into
secretory cells.
iBET-151 reversibly promotes proliferation and inhibits
secretory differentiation
The cell fate conversion of intestinal epithelial cells in vivo is highly
dynamic, involving various simultaneous cellular events, including
self-renewal, differentiation, and dedifferentiation. We next asked
Fig. 2 | scRNA-seq analysis reveals cellular diversity and cell fate dynamics in
TpC-organoids. a UMAP plot showing clustering of cells from scRNA-seq analysis
of TpC organoids. Cluster labels indicate cell types. LGR5.high and LGR5.low indi-
cate intestinal stem cells with different levels of LGR5 expression; TA1, transit-
amplifying cell type 1; TA2, transit-amplifying cell type 2; Sec Pre, Secretory Pre-
cursor;Sec Pre2, SecretoryPrecursor Type 2; EC, enterocyte;EEC, enteroendocrine
cell. bProportionof each cell type from scRNA-seq sample in a.cDotplotshowing
expression and percentage of cellsexpressing cell type-specic markers in clusters
from scRNA-seq analysis. Dot size and color indicate normalized gene expression
level. dUMAP plots showing expression of cell type-specic markers in TpC
organoids. Color intensity indicates normalized gene expression level. eMA plot
showing differentially expressed genes between Paneth cells and LGR5-high cells.
fHeatmap showing predicted PROGENy pathway activity for each cell cluster.
gUMAP plot showing subset analysis of secretory cells along with connected cell
clusters. hPie chart showing the proportion of each secretory cell type. Total
percentages of all secretory cell types equal 100%. iDot plot showing expression
and percentage of cells expressing cell type-specicmarkers in clustersshown in g.
Dot sizeand color indicate normalized geneexpressionlevel. jUMAP plotsshowing
expression of cell type-specic markers in the secretory subset. Color intensity
indicates normalized gene expression level. kUMAP plots highlighting EEC cells
and depicting expression of EEC subtype-specic markers. Color intensity indicates
normalized gene expression level. lPAGA trajectory analysis depicting cell con-
nectivity. Circle size indicates cell number,and line thickness indicates connectivity
strength between cell clusters. mRNA velocity analysis using Dynamo showing
inferred transition direction between cell states. nPie charts (top) showing pro-
portions of indicated cell types among total cells, and bar charts (bottom) showing
the composition of indicated cell types from scRNA-seq analysis of ES, IF, IL, TpC
organoids, and crypt.
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved
whether these processes could be similarly regulated in our TpC
organoids, which exhibit a balanced self-renewal and differentiation.
To this end, we furtherevaluated theeffects of small moleculepathway
regulators based on the TpC condition (Fig. 4a). We discovered that
iBET-151 (iBET or I), a BET bromodomain inhibitor, effectively
enhanced cell proliferation while decreasing the number of LGR5-
mNeonGreen cells (Fig. 4bf). Concurrently, we observed reduced
differentiation of secretory cells, including Paneth cells, goblet cells,
and EECs (Fig. 4e, f and Supplementary Fig. 16a, b). Notably, Paneth
cellswerenearlyabsentintheorganoids(Fig.4e, f and Supplementary
Fig. 16b, d). Additionally, the presence of iBET induced a distinct
morphological change in the crypt domain of the organoids, resulting
in a more homogeneous cell population rather than intermingled
Paneth and ISC-like cells (Supplementary Fig. 16c). These ndings
b e
lm
p
no r
k
q
d
g
PC-AST- -pVc
LYZ DAPI
CHGA DAPI MUC2 DAPI
24 days
12 days 12 days
TpC
TpC
TpC-CP
EdU
TpC
EdU
-CP
Count
mNeonGreen
LGR5 high
TpC-CP
TpC
100101102103104105
300
200
100
0
c
j
i
a
Basal
+TSA
+pVc
+CP
+TSA.pVc
+TSA.CP
+pVc.CP
+TSA.pVc.CP
0
2
4
6
8
LGR5-mNeonGreen (%)
P < 0.0001
P < 0.0001
P < 0.0001
P<0.0001
P < 0.0001
P < 0.0001
P = 0.4162
Basal
+TSA
+pVc
+CP
+TSA.pVc
+TSA.CP
+pVc.CP
+TSA.pVc.CP
0
500
1000
1500
Relative mean intensity
P=0.0013
P < 0.0001
P = 0.0467
P < 0.0001
P < 0.0001
P < 0.0001
P = 0.9962
Basal
+TSA
+pVc
+CP
+TSA.pVc
+TSA.CP
+pVc.CP
+TSA.pVc.CP
0
10
20
30
40
Colony forming efficiency (%)
P < 0.0001
P<0.0001
P < 0.0001
P < 0.0001
P = 0.9990
P = 0.9988
P = 0.9500
Basal
+TSA
+pVc
+CP
+TSA.pVc
+TSA.CP
+pVc.CP
+TSA.pVc.CP
0
5
10
15
Number of cells(×10
5
)
P < 0.0001
P<0.0001
P < 0.0001
P < 0.0001
P > 0.9999
P = 0.2032
P = 0.3453
LYZ MUC2 CHGA
0
10
20
30
Positive cells (%)
TpC
-TSA
-pVc
-CP
P = 0.0014
P = 0.0029
P = 0.0006
P = 0.0045
P = 0.0004
=
P06
710.
0
139.
0
=P
P=62
5
9
.0
f
h
0.0
0.5
1.0
1.5
2.0
P = 0.0076
P<0.0001
LYZ
P = 0.0048
P = 0.1988
P = 0.3322
P > 0.9999
P = 0.1166
LGR5 SMOC2
0
1
2
3
4
5
P < 0.0001
P<0.0001
P = 0.0125
P<0.0001
P < 0.0001
P<0.0001
P<0.0001
P = 0.0026
P = 0.0001
P = 0.9979
P = 0.9993
P = 0.0002
P = 0.3706
P = 0.2324
Basal
+TSA
+pVc
+CP
+TSA.pVc
+TSA.CP
+pVc.CP
+TSA.pVc.CP
DEFA5 DEFA6
0
5
10
15
Relative Expression
P<0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P<0.0001
P<0.0001
P = 0.0979
P = 0.9792
P = 0.9998
P = 0.0022
P = 0.5681
P = 0.2272
P = 0.0020
P = 0.0016
P =0.0058
P =0.0038
EdU
LYZ
MUC2
CHGA
0
5
10
15
20
25
Positive cells (%)
TpC
-CP
TpC
-TSA
-TSA
+KCC
0
1
2
3
4
LGR5-mNeonGreen (%)
P<0.0001
P<0.0001
pC
+TSA
+KCC
pC
+TS A
+KCC
0
2
4
6
8
LGR5-mNeonGreen (%)
P<0.0001
P=0.0113
P<0.0001
P<0.0001
P<0.0001
WT MBD3-KD
pC +TSA +KCC
MBD3-KD WT
LGR5
LGR5
Excluding factors from TpC condition
LGR5-mNeonGreen Brightfield
TpC -CP
Withdrawing CP from TpC organoids
LYZ
EdU LYZ DAPI
LYZ
EdU LYZ DAPI
TpC -CP
MUC2
MUC2 DAPI
MUC2
MUC2 DAPI
TpC -CP
CHGA DAPI
CHGA
CHGA DAPI
CHGA
TpC -CP
Withdrawing CP from TpC organoids: Cell differentiation
TpC
-CP
0
100
200
300
Relative mean intensity
P<0.0001
TpC
-CP
0
1
2
3
4
LGR5-mNeonGreen (%)
P<0.0001
Withdrawing CP from TpC organoids
Replacing TSA with MBD2/3 inhibition
Basal +TSA +pVc +CP +TSA.pVc +TSA.CP +pVc.CP +TSA.pVc.CP (TpC)
LGR5-mNeonGreen Brightfield
Adding factors to Basal condition for 1 week from single cells
P=.
025
2
4
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
imply that the TpCI combination maintains the organoids in a more
progenitor-like state, partially by inhibiting differentiation towards
secretory cell types.
To examine the reversibility of iBETs effects and assess the
functionality of TpCI-cultured cells, we removed iBET from TpCI-
cultured organoids (Supplementary Fig. 16e). After seven days of
continued culture in TpC conditions, the LGR5-mNeonGreen expres-
sion increased to levels comparable to those observed in TpC orga-
noids (Supplementary Fig. 16f, g), accompanied by the resurgence of
secretory cells in the organoids (Supplementary Fig. 16h, i).
To investigate the impact of iBET on established organoids, we
introduced iBET to TpC organoids (Supplementary Fig. 16j). After
culturing for 12 days in the TpCI condition, we observed a notable
enhancement in organoid proliferation as evidenced by the increased
organoid size and the presence of EdU-positive proliferating cells
(Supplementary Fig. 16j-k). In contrast, secretory cell differentiation
was reduced in the organoids (Supplementary Fig. 16j-k), consistent
with observations in organoids derived from single intestinal cells.
These ndings suggest that the effects of iBET are reversible, and the
cell fate of cells in TpC or TpCI organoids is dynamic, adjusting in
response to different culture conditions.
To further elucidate the cellular diversity and cell fate trajectory
within TpCI-cultured organoids, we performed single-cell RNA
sequencing (scRNA-seq). Celltype annotation revealed the presenceof
most cell types found in TpC organoids, with the notable exception of
Paneth cells (Fig. 4g). This absence was further conrmed by the lack of
expression of Paneth cell markers (Fig. 4h, i). Cell composition analysis
indicated a lower abundance of secretory cells, such as enteroendo-
crine cells (EECs) and secretory precursor cells, while the abundance of
goblet cells remained similar. However, there was a notable increase in
enterocyte lineage cells and transient amplifying (TA) cells (Fig. 4j).
This observation aligns with the cellular phenotype of TpCI organoids,
which exhibit increased proliferation and reduced differentiation into
secretory cells. Cell type priority analysis using Augur indicated that
LGR5-high and early enterocyte (EC) cells were most affected by iBET
treatment (Fig. 4k). This suggests that iBET treatment alters the cell
differentiation trajectory, shifting it from secretory differentiation
towards the enterocyte lineage. Further trajectory and MetaCell ana-
lysis revealed a major cell differentiation pathway from TA cells to
enterocytes, mediatedby LGR5 cells (Fig. 4l, m). This data supports the
hypothesis that iBET treatment promotes the proliferation of enter-
ocyte lineage cells at the expense of secretory cell differentiation.
Controlled lineage shifts in organoids via dened niche signals
Our optimized combination of chemical modulators effectively sus-
tained a balance between stem cell self-renewal and differentiation in
the organoids, as well as the controlled shift of this equilibrium deci-
sively towards enhanced proliferation. Going forward, we aimed to
identify conditions that can selectively and unidirectionally shift this
equilibrium towards specic differentiated cell types, allowing us to
generate organoids enriched in Paneth, goblet, enteroendocrine, or
enterocyte cells. Although several protocols have been reported to
induce Paneth cell or enteroendocrine cell differentiation, such as
using IL-22 for Paneth cells11 or modulating the endocannabinoid
receptor, c-Jun N-terminal kinase (JNK) or Forkhead box O1 (FOXO1)
pathways for enteroendocrine cells32, we aimed to induce differentia-
tion by recapitulating the physiologic cell fate dynamics directed by
in vivo niche signals such as Wnt, Notch and BMP signaling.
We systematically screened various combinations of Wnt, Notch,
BMP, and EGFR pathway modulatorsin a stepwise fashion (Fig. 5a). We
found that the effects of Wnt and Notch signaling on mouse ISCs also
applied to human cells. Specically, inhibiting Notch signaling with
DAPT promoted differentiation towards secretory cells3335. Additional
modulation of Wnt activity further directed differentiation towards
Paneth cells, goblet cells, or enteroendocrine cells. Sustained Wnt
activation in the presence of DAPT enhanced Paneth cell differentia-
tion. Further addition of IL-22 promoted Paneth cell differentiation
(Supplementary Fig. 17a), resulting in a 15%enrichment of Paneth cells
after a rapid 3-day induction protocol (Fig. 5b, f). In contrast, we found
that an initial step of Wnt activation and Notch inhibition followed by
Wnt inhibition and sustained Notch inhibition was required to enhance
goblet cell and enteroendocrine cell differentiation. This may corre-
spond to a secretory precursor stage in vivo. Moreover, switching BMP
signaling from inhibition to activation was essential for goblet cell
induction (Supplementary Fig. 17b), leading to nearly 50% goblet cell
differentiation efciency from TpC organoids (Fig. 5c, g). While CP
mildly inhibited enteroendocrine cell generation in TpC organoids,
removing CP did not further enhance its differentiation. Therefore, we
included CP and the EGFR inhibitor Getinib in the protocol to prevent
Paneth cell or goblet cell differentiation and induce enteroendocrine
cell differentiation36. Under these conditions, CHGA-positive enter-
oendocrine cells comprised 17% of organoid cells (Fig. 5d, h). The
duration of step 1 and step 2, primarily distinguished by the presence
of Wnt signaling, signicantly affected the differentiation efciency of
goblet cells and enteroendocrine cells (Supplementary Fig. 17b, c),
similar to the differentiation of secretory cells in mouse organoid
cells37. Furthermore, the combination of IWR-1, SJ000291942, and VPA
rapidly and efciently induced enterocyte differentiation in three days
(Fig. 5e). Under these conditions, Paneth cells, goblet cells, and
enteroendocrine cells were not enriched (Fig. 5i). Removing any single
factor reduced enterocyte differentiation (Supplementary Fig. 17d).
We further tested these differentiation protocols on TpCI orga-
noids, which are enriched for enterocyte lineage cells (Fig. 5j). As
anticipated, these protocols induced differentiation with higher ef-
ciency of EC cells in TpCI organoids compared to TpC organoids
(Fig. 5k, l), while relatively lower differentiation efciency of Paneth
cell and EEC than TpC organoids. We also tested the Paneth cell dif-
ferentiation protocol on IF organoids and detected a relatively low
Fig. 3 | The combination of TSA, pVc, and CP induces enhanced stemness and
increased cellular diversity in organoids. a Brighteld and uorescence images
of organoids cultured for 1 week in indicated conditions. Representative images
from three independent experiments. b, c Quantication of the percentage of
LGR5-mNeonGreen cells (b) and relative uorescence intensity (c)inorganoids
cultured in indicated conditions, n= 4 samples. Colony forming efciency (d)and
the number of cells (e) of organoidscultured in indicated conditions,n=4samples.
fRT-qPCR quantication of cell markers expression levels, n=3samples.
gRepresentative confocal images of LYZ, MUC2, and CHGA in organoids cultured
in indicated conditions. Representative images from three independent experi-
ments. hProportions of secretory lineages in organoids cultured in indicated
conditions, n=4samples. bfand h, one-way ANOVA with Dunnetts multiple
comparisons test; data are presented as mean ± SD. iSchematic of experiments in
(jo). jConfocal images of EdU staining of organoids. kQuantication of the per-
centageof LGR5-mNeonGreen cellsand relative uorescenceintensity in organoids
cultured under the indicated conditions, n=3samples.lBrighteld and uores-
cence images of organoids cultured in indicated conditions. mEdU and immuno-
uorescence staining of CHGA, MUC2, and LYZ in organoid cultures as indicated.
EdU was added for 1 hour before staining. Representative images of (j,l,m)were
from three independent experiments. nFACS gating strategy of LGR5-
mNeonGreen cells. oQuantication of EdU, LYZ, MUC2 and CHGA positive cell
percentage as in (m), n=4samples. kand o, two-tailed unpaired t-test; data are
presented as mean ± SD. pQuantication of the percentage of LGR5-mNeonGreen
cells in organoids cultured under the indicated conditions, n= 6 samples. qLGR5-
mNeonGreen uorescence images of organoids cultured in indicated conditions.
Representativeimages from four independentexperiments. rProportions of LGR5-
mNeonGreen cells in organoids cultured in conditions as in (q), n=4samples.
(pand r), one-way ANOVA with Dunnetts multiple comparisons test; data are
presented as mean ± SD. Scalebars, (aand l), 200 μm; (g,j,mand q), 50 μm. Source
data are provided as a Source Data le.
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved
proportion of Paneth cells after a 3-days induction (Supplementary
Fig. 17e). Additionally, we applied a previously published M cell dif-
ferentiation protocol for mouse intestinal cells38,39 to human TpC
organoids and observed high-efciency differentiation (Fig. 5mand
Supplementary Fig. 18a, b).
These results demonstrate that stem cells in TpC organoids
remain multipotent. The equilibrium state of TpC organoids can be
selectively shifted through dened signaling toward the major func-
tional cell types of the human intestine (Supplementary Fig. 19a). By
integrating our differentiation data with single-cell trajectory analysis,
we propose a unied model of cell fate transitions in the human
intestinal epithelium (Supplementary Fig. 19b). In this model, spatio-
temporal gradients of Wnt, BMP, and Notch signaling direct ISC self-
renewal and differentiation. ISC self-renewal at the crypt base results
from high Wnt, low BMP, and activated Notch signaling and can be
further enhanced by factors such as pVc and CP. Downregulation of
Notch signaling at the crypt base gives riseto secretory precursors in a
high Wnt, low BMP niche. Depending on the Wnt and BMP levels, these
a
dcb
ef
8000 single cells
Analyze
21 days
TpC
21 days
TpC + iBET
CpTTEBi+CpT
Single
intestinal cells
-iBET-151
Reversible
Proliferation
Early EC
Differentiation
Proliferation
LGR5+
Differentiation
Goblet cells
ISCs
Paneth cells
Enteroendocrine cells
Enterocytes
Tuft cells
TA/Early ECs
Screening
iBET-151
TpC +
TEBi+CpT
DEFA5 EdU DEFA5 EdU DAPI
CHGA DAPI
MUC2 DAPI
LYZ DAPI
DEFA5 EdU DEFA5 EdU DAPI
CHGA DAPIMUC2 DAPILYZ DAPI
Brightfield
LGR5-mNeonGreen
TpC +iBET
TpC
+iBET
0
2
4
6
LGR5-mNeonGreen (%)
P < 0.0001
TpC
+iBET
0
200
400
600
800
1000
Relative mean intensity
P < 0.0001
EdU DEFA5 MUC2 CHGA
0
10
20
30
40
Positive cells (%)
TpC
+iBET
P = 0.0342
P=0.0015
P = 0.0215
P = 0.0201
h
lig
k
23
16
3
4
4
29
2
2
11
6
36
10
2
5
4
18
12
14
0
25
50
75
100
TpC +iBET
Proportion of cells (%)
Cell type
LGR5.high
LGR5.low
TA1
TA2
Early EC
EC
Sec Pre
Sec Pre2
Goblet
EEC
Paneth
Tuf t
Cell composition change
following iBET treatment
FGFBP1
FABP2
MUC2LGR5 DEFA5
CHGAOLFM4 DEFA6
20 40 60 80100
Percent expressed
01
Mean expression
ISC
TA1
TA2
Sec Pre
Goblet
Paneth
EEC
Tuft
EC
LGR5
OLFM4
ASCL2
HES1
SMOC2
PCNA
HELLS
MCM6
MKI67
TOP2A
HES6
DLL1
DLL4
MUC2
SPINK4
FCGBP
DEFA6
DEFA5
PRSS2
CHGA
NEUROD1
CHGB
NEUROG3
ALOX5
AVIL
RGS13
FGFBP1
FABP1
KRT20
MTTP
SLC5A1
ACE2
ALDOB
APOA4
RBP2
ALPI
PCK1
LGR5.high
LGR5.low
TA1
TA2
Sec Pre
Goblet
EEC
Early EC
EC
EC
umap1
umap2
Cell Type
EEC
Early EC
Goblet
LGR5.high
LGR5.low
Sec Pre
TA1
TA2
umap1
umap2
Pseudotime
0.4
0.8
MetaCell analysis of iBET
treated organoids with SEACells
0.5 0.6
Augur score by cell type
EEC
EC
LGR5.low
Goblet
TA1
Sec Pre
TA2
Early EC
LGR5.high
Cell type responsiveness
to iBET quantified by Augur
j
m
MKI67
OLFM4
HES1
LGR5
FGFBP1
FABP 1
FXYD3
KRT20
PCK1
ALDOB
SLC11A2
MTTP
SLC5A1
ALPI
APOA4
ACE2
RBP2
GSTA1
Marker gene expression along pseudotime
max
min
Early EC
EC
EEC
Goblet
LGR5.high
LGR5.low
Sec Pre
TA1
TA2
umap1
umap2
UMAP plot of iBET treated organoids
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved
cells further differentiate into distinct secretory cell types. In contrast,
loss of Wnt reduces ISC self-renewal and promotes their differentiation
into enterocytes. During this enterocyte differentiation process, par-
tially differentiated cells can further differentiate into secretory cells,
especially goblet cells, upon Notch inactivation. This equilibrium can
be further shifted by chemical or genetic perturbations. For instance,
we showed that NuRD complex inhibition and BET inhibition have
distinct effects on cell fate trajectory and consequently on cellular
composition in the organoids.
Discussion
In this study, we demonstrate the ability to mimic complex cell fate
dynamics observed in vivo by using growth factors and small mole-
cules within an organoid system. By precisely controlling cell fate
dynamics, our method fosters a balanced state of self-renewal and
differentiation in organoids, which in turn allows for parallel pro-
liferation and enhanced cellular diversity. Furthermore, by manip-
ulating combinations of niche factors, we induced cell fate shifts
towards different differentiation directions, signicantly altering the
cellular composition of the organoid.
Maintaining balanced self-renewal and differentiation of adult
stem cells is crucial for tissue homeostasis in vivo. However, achieving
this balance in vitro has been challenging in organoid culture, primarily
due to the absence of signaling gradients regulating localized self-
renewal and differentiation within specicniches
12,40,41.Furthermore,
while progenitor cells within organoids are sufcient for driving both
proliferation and differentiation, their limited plasticity and stemness
ultimately restrict the diversity of cell types in organoids. In this study,
we hypothesize that maximal cellular diversity in organoids can be
more effectively achieved by inducing enhanced stemness within
organoids, thereby unlocking their potency and potential for multi-
directional differentiation rather than relying on lineage-restricted
progenitors or directly driving differentiation. We demonstrated that
this regress to progressstrategy applies to human intestinal orga-
noids. By modulating the combinations of small molecules, we
enhanced the stemness of intestinal stem/progenitor cells without
hindering their differentiation, thereby achieving a balanced self-
renewal and differentiation within the organoids. This approach led to
both rapid growth and increasedcellular diversity,offering advantages
over existing protocols. The abundance of LGR5+stem cells enables
efcient organoid establishment and rapid expansion and, impor-
tantly, endows the capacity to generate a diverse range of intestinal
cell types. Our protocol produced Paneth cells, enteroendocrine cells,
goblet cells, tuft cells, and mature enterocytes without requiring a
separate differentiation step. This substantially improves previous
methods and provides an optimized human intestinal organoid system
for applications such as drug development and disease modeling.
We have veried the specic roles of the three key components,
TSA, pVc, and CP673451, in the TpC system and demonstrated that
TSA, as an HDAC inhibitor, exerts its effects through the NuRD
complex. Further investigation into the epigenetic mechanisms of
HDAC inhibitors, using tools such as ATAC-seq, would provide
valuable insights and represent a promising direction for future
research.
While a signaling pathway regulation network for mouse ISC
differentiation to specic cell types like Paneth, goblet, enter-
oendocrine, enterocytes, M cells, and Tuft cells has been
established22,36,42,43, developing similar networks for human intest-
inal epithelial cells has been challenging due to the difculty in
maintaining human ISCs. Although obtaining high-purity hISC cul-
tures has been difcult, we established an organoi d system enriched
in LGR5+cells. Additionally, we showed that iBET-151 reversibly
shifted cell fate from secretory cell differentiation to the enterocyte
lineage while maintaining cells in a highly proliferative state, pro-
viding a tool to dissect the regulatory network for major cell types in
the human intestinal epithelium. We found similar signaling
mechanisms regulate Paneth, enteroendocrine, goblet cell, and
enterocyte differentiation in both human and mouse intestinal
epithelial cells, involving combinations and timing of Wnt, Notch,
BMP, and MAPK pathways. Paneth cells can be obtained from
human cells using high Wnt and Notch inhibitor s, as in mouse ISCs22.
We found an intermediate, high Wnt, and low BMP step upon Notch
inhibition effectively increased secretory cell differentiation,
representing secretory precursor differentiation at the crypt base
in vivo upon Notch inac tivation. These results suggest that basic cell
fate regulation mechanisms are conserved in human and mouse
intestinal cells, relying on niche signaling combinations.
While our scRNA-seq analysis and experiments suggest poten-
tial cellular plasticity in our organoid system, conclusively demon-
strating direct transdifferentiation between cell types requires
additional validation. Future studies using advanced lineage tracing
techniques, such as CRISPR-mediated reporter knock-in organoids
and unbiased cell barcoding approaches, will be essential to de-
nitively map cell fate transitions and better understand the
dynamics of cellular plasticity in human intestinal organoids. These
studies will help elucidate the precise mechanisms underlying dif-
ferentiation, dedifferentiation, and potential transdifferentiation
events observed in our system.
Beyond these molecular and cellular dynamics, another key
challenge remains in recreating the complex tissue architecture of the
intestinal epithelium. Although our TpC system has increased cellular
diversity in the organoids, it has not yet achieved the correct spatial
structure and cell distribution as seen in vivo. Ultimately, creating an
in vitro model that fully emulates in vivo physiology requires repro-
ducing the capacity of stem cells to respond to complex spatio-
temporal cues governing their self-renewal and differentiation, where
a homogenous culture environment may limit the maturation of dif-
ferentiated cells. Thus,we envision that articially generating signaling
Fig. 4 | iBET-151 reversibly promotes proliferation and inhibits secretory dif-
ferentiation. a Schematic of the screening strategy for modulating early EC
enrichment in organoids. bSchematic of organoid culture conditions for experi-
ments in (cf). cRepresentative brighteld and uorescence images of organoids
cultured in indicated conditions. Representative images from three independent
experiments. Scale bars, 200 μm. dProportion of LGR5-mNeonGreen cells in
organoids shown in (c). n= 5 samples. Two-tailed unpaired t-test; data are pre-
sented as me an± S D. eEdU and immunouorescence staining of Paneth cells (LYZ
and DEFA5), goblet cells (MUC2), and enteroendocrine cells (CHGA) in organoids
cultured as in (b). Representative images from three independent experiments.
Scale bars,50 μm. fQuantication of positive staining as in (e).n= 3 samples. Two-
tailed unpaired t-test; data are presented as mean ± SD. gUMAP plot showing
clustering of cells from scRNA-seq analysis of TpCI (TpC+iBET) organoids. Cluster
labels indicate distinct cell types. hUMAP plots displaying the expression levels of
cell type-specic markers (LGR5,MUC2,DEFA5,FGFBP1,OLFM4,CHGA,DEFA6,
FABP2) in TpCI organoids. Color intensity indicates normalized gene expression
levels. iDot plot illustrating the expression and percentage of cells expressing cell
type-specic markers across the clusters identied in (g). Dot size represents the
percentage of cells expressing the marker, while color intensity indicates normal-
ized geneexpression levels. jBar charts depicting the composition of indicated cell
types from scRNA-seq analysis, comparing TpCI organoids with TpC organoids.
Proportions of cell types are shown as percentages. kCell type responsiveness to
iBET treatment quantied by Augur, comparing the TpCI dataset with the TpC
dataset. Augur scores for each cell type are displayed, indicating the degree of
responsiveness to iBET treatment. lUMAP plot demonstrating pseudotime analysis
and cell type labeling of MetaCells in the iBET dataset. Pseudotime was calculated
usingthe Stavia package,and MetaCells wereidentied usingthe SEACellspackage.
mHeatmap of cell type-specic marker gene expression along the pseudotime
trajectory as shown in (l).The color bar indicatescell type labels consistent with (l).
Source data are provided as a Source Data le.
Article https://doi.org/10.1038/s41467-024-55567-2
Nature Communications | (2025) 16:315 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
gradients through bioengineering approaches could further improve
the delity of organoid models.
Methods
The research reported here complies with all relevant ethical regula-
tions and guidelines. Small intestine tissue biopsies used in this study
were obtained from patients who provided written informed consent
under the ethical committee of Shanghai East Hospital (approval
number: 2023-064).
Genetic engineering of human small intestinal organoids
For the construction of a uorescent reporter, an LGR5-P2A-
mNeonGreen knock-in reporter was introduced into the last exon of
LGR5 via electroporation, using previously published sgRNA (Sup-
3 days
TpC
Wnt -
Notch +
BMP +
hSIO Enterocyte
enriched
3 days2 days
TpC
Wnt +
Notch -
BMP -
Wnt -
Notch -
BMP -
EGFR -
hSIO EEC
enriched
2 days 3 days
TpC
Wnt +
Notch -
BMP -
Wnt -
Notch -
BMP +
hSIO Goblet
enriched
3 days
TpC
Wnt +
Notch -
BMP -
IL-22
hSIO Paneth
enriched
b
a
ed
ihgf
j
l
km
c
CHIR99021
VPA
SJ000291942
EGF
Wnt
Notch
BMP
EGFR
IWR-1
DAPT
DMH1
Gefitinib
Stepwise
Combination
Screening
Pathway modulators
Expansion
TpC
Paneth cell enriched
EEC enriched
Goblet cell enriched
Enterocyte enriched
Single cells hSIO
ALPI
LYZ DAPI
CHGA DAPI
LYZ
MUC2
CHGA
ALPI
LYZ
MUC2
CHGA
ALPI
LYZ DAPI
MUC2 DAPI
CHGA DAPI
ALPI
LYZ
MUC2
CHGA
ALPI
LYZ DAPI
MUC2 DAPI
CHGA DAPI
ALPI
LYZ DAPI MUC2 DAPI CHGA DAPI
Panet