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Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture – Nature Communications

Expansion of hepatocyte-derived cells in culture

We crossed mice expressing an inducible form of Cre recombinase (CreERT2) from the albumin (Alb) genomic locus [Alb-CreERT2 mice20] with R26RYFP/YFP reporter mice21. In the double-mutant mice, administration of tamoxifen (TM) permanently marked Alb-positive hepatocytes and enabled tracing of their fates.

To observe hepatocytes in culture, we isolated them from the livers of TM-treated Alb-CreERT2;R26RYFP/+ mice and cultured them by serial passaging. Three hours after their initial plating, co-immunofluorescence analyses revealed that all YFP-positive cells expressed both Alb and Hnf4α, but YFP was not detected with the cholangiocyte marker cytokeratin 19 (CK19; also known as Krt19) (Fig. 1a, b). Thus, we have developed an experimental system for tracing the fate of hepatocytes in culture that involves highly specific isolation of hepatocytes and their heritable labeling with YFP.

Fig. 1: Propagation of hepatocyte-derived cells in monolayer culture.
figure 1

a Hepatocytes isolated from adult mouse livers were stained with antibodies against YFP and Alb, Hnf4α, or CK19, 3 h after plating. DNA was stained with DAPI. The graph shows the percentages of YFP+, Alb+, Hnf4α+, and CK19+ cells in the DAPI+ population and the percentages of Alb+, Hnf4α+, and CK19+ cells in the YFP+ population. Data represent the mean ± SEM (n= 5 independent experiments). Source data are provided as a Source Data file. b Representative micrographs of isolated hepatocytes expressing YFP and Alb, Hnf4α, or CK19. DNA was stained with DAPI. c Representative fluorescence micrographs and morphologies of YFP+ hepatocytes 3 h, 4 days (d), 1 week (w), and 2 w after plating and at passage 4 (P4). Scale bars, 100 μm.

In monolayer culture, the morphology of some hepatocytes shifted from round to spindle-shaped within 4 days after plating; these cells continued to proliferate and gradually became small epithelial cells after several weeks of culture (Fig. 1c). These YFP-positive hepatocyte-derived cells could be maintained through serial passaging (Fig. 1c). Two independent experiments demonstrated that YFP was expressed in more than 99% of cultured cells derived from the liver of TM-treated Alb-CreERT2;R26RYFP/+ mice, indicating that their hepatocyte progenies can proliferate and expand in long-term culture (Supplementary Fig. 1a, b). Moreover, similar to hepatocytes isolated form Alb-CreERT2;R26RYFP/+ mice, those from untreated wild-type mice were also capable of propagating in culture (Supplementary Fig. 1c). Thus, these cells were named dedifferentiated hepatocytes (dediHeps).

dediHeps have a hybrid E/M phenotype

During conversion of primary hepatocytes to dediHeps, the expression of Alb and the epithelial cell marker E-cadherin (E-cad; also known as Cdh1) was maintained (Fig. 2a). Along with Alb and E-cad, CK19 and the mesenchymal cell marker Vimentin (Vim) were expressed in spindle-shaped hepatocyte-derived cells 4 days after plating; their expression was maintained in long-term dediHep cultures (Fig. 2a). Confocal microscopy clearly demonstrated that Alb and E-cad were co-expressed with CK19 and Vim, respectively, in dediHeps (Fig. 2b). These findings suggest that primary hepatocytes adopt a hybrid E/M phenotype during the early stages of monolayer culture, with this phenotype maintained during and after the transition from hepatocyte to dediHep. Expression levels of Cdh1 and the epithelial cell markers claudin-3 (Cldn3) and occludin (Ocln) were not changed, but expression of Vim and the mesenchymal cell markers S100 calcium binding protein A7A (S100a7a) and laminin B1 (Lamb1) increased, in cultured dediHeps relative to expression in hepatocytes freshly isolated from adult mouse livers (Fig. 2c). In addition, expression of the proliferation marker Ki67 (also known as MKi67) and the cholangiocyte/progenitor marker prominin-1 (Prom1; also known as CD133) was increased and expression of several genes encoding liver enzymes was decreased in dediHeps relative to their expression in hepatocytes, while both dediHeps and hepatocytes expressed similar levels of transcription factors involved in hepatocyte differentiation, including Hnf4α and Hnf1α (Fig. 2c). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that genes associated with the cell cycle and metabolic pathways were enriched among those that were up- and down-regulated, respectively (Fig. 2d). Sequential gene expression analyses during hepatocyte-to-dediHep conversion revealed that the expression of genes encoding liver enzymes began to decrease after 1 day in monolayer culture, followed by subsequent increases in the expression of MKi67, Krt19, Prom1, and mesenchymal cell markers (Fig. 2e). Taken together, our data demonstrate that dediHeps maintain a subset of key characteristics of hepatocytes and epithelial cells, but lose function and acquire a partial mesenchymal phenotype.

Fig. 2: dediHeps exhibit a hybrid E/M phenotype.
figure 2

a Co-immunofluorescence staining (YFP with Alb, CK19, E-cad, or Vim) of hepatocytes in monolayer culture 3 h, 4 days (d), 1 week (w), and 2 w after plating and at passage 4 (P4). b Representative confocal microscopic micrographs of dediHeps that are immunoreactive for Alb with CK19 and for E-cad with Vim at P4. DNA was stained with DAPI. Scale bars, 100 μm (a) and 20 μm (b). c Volcano plot depicting differences in gene expression between dediHeps and hepatocytes. Each dot represents one gene. Blue and red dots represent down- and up-regulated genes, respectively, in dediHeps. Gray dots represent genes that are not significantly differentially expressed. d Top five most significantly enriched pathways associated with down- and up-regulated genes in dediHeps compared to hepatocytes, identified by KEGG pathway enrichment analysis. e Heatmap image from CEL-seq2 data showing the change in expression of the indicated genes during conversion of freshly isolated hepatocytes (FH) to dediHeps in monolayer culture 3 h, 1 d, 2 d, 4 d, 1 w, and 2 w after plating and at P10. Statistical difference was determined by quasi-likelihood F-test (c) or Fisher’s exact test (d).

dediHeps re-differentiate into hepatocytes and reconstitute liver tissues after transplantation into injured livers

To investigate re-differentiation potential, we developed dediHep aggregate cultures, as aggregation has been shown to facilitate the maturation of immature hepatocyte-like cells derived from pluripotent stem cells22 or directly induced from fibroblasts23. Under 3D culture conditions, dediHeps gradually assembled to form uniform aggregates (Fig. 3a), expressing Alb and E-cad, but neither Vim nor Ki67 (Fig. 3b). To assess the hepatic maturation of dediHep aggregates, we performed gene expression analyses in them, with monolayer controls, and conduced KEGG pathway enrichment analyses. The expression of genes encoding liver enzymes was increased, but expression of MKi67, Krt19, Prom1, and mesenchymal cell markers was decreased, in aggregates (Fig. 3c). The changes in gene expression were promptly induced starting from 1 day of 3D culture (Supplementary Fig. 2). Meanwhile, the expression levels of Cdh1, Cldn3, Ocln, Hnf4α, and Hnf1α were almost unchanged between aggregates and monolayers (Fig. 3c). KEGG pathway enrichment analyses revealed that genes differentially expressed in dediHep aggregates versus monolayers were significantly enriched for those associated with metabolic pathways (up-regulated) and cell cycle (down-regulated) (Fig. 3d). The hepatic maturation of dediHep aggregates could also be induced from dediHeps propagated in long-term monolayer culture (Supplementary Fig. 3).

Fig. 3: Re-differentiation of dediHeps into functional and transplantable hepatocytes in 3D culture.
figure 3

a Representative morphology of a dediHep aggregate at the presented hours (h) and days (d) after initiation of 3D culture. Inset, the enlarged micrograph of aggregate. b Co-immunofluorescence staining (E-cad with Alb, Vim, or Ki67) of dediHep aggregates 5 days after initiation of 3D culture. c Volcano plot depicting differences in gene expression between dediHep aggregates and dediHep monolayer cultures. Each dot represents one gene. Blue and red dots represent down- and up-regulated genes, respectively, in dediHep aggregates. Gray dots represent genes that are not significantly differentially expressed. d Top five most significantly enriched pathways associated with down- or up-regulated genes in dediHep aggregates compared to dediHep monolayer cultures, identified by KEGG pathway enrichment analysis. e Immunohistochemical staining of Fah and co-immunofluorescence staining of YFP with Fah on liver sections of Fah-/- mice 3 months after transplantation of cells dissociated from dediHep aggregates derived from TM-treated Alb-CreERT2;R26RYFP/+ mice. f A representative micrograph of Fah+ cells on liver sections of Fah-/- mice 3 months after transplantation of hepatocytes freshly isolated from the liver of adult wild-type mice. Graph at right depicts percentages of Fah+ cells in CK8/18+ epithelial cells on liver sections of Fah-/- mice 3 months after transplantation of cells dissociated from dediHep aggregates or hepatocytes. Data represent the mean ± SEM (n= 3 independent experiments). DNA was stained with DAPI. Scale bars, 200 μm (a, e, and f) and 20 μm (b). Statistical difference was determined by quasi-likelihood F-test (c), Fisher’s exact test (d), or two-sided Student’s t test (f). Source data are provided as a Source Data file.

In order to determine whether liver parenchyma could be reconstituted by dediHep-derived, re-differentiated hepatocytes, we intrasplenically injected dissociated aggregates from TM-treated Alb-CreERT2;R26RYFP/+ mice into the livers of fumarylacetoacetate hydrolase (Fah)-deficient (Fah-/-) recipient mice, which is a mouse model of hereditary tyrosinaemia type I24. Fah-/- livers are severely damaged without the provision of 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC); therefore, these mice can be used as recipients to analyze hepatocyte differentiation potential and hepatic tissue reconstitution. Three months after transplantation, dediHep-derived, YFP-positive cells reconstituted hepatic tissues of Fah-/- recipients as Fah-positive hepatocytes and were morphologically indistinguishable from normal hepatocytes (Fig. 3e). These dediHep-derived hepatocytes did not express the mesenchymal cell markers Vim and α-smooth muscle actin (αSMA) (Supplementary Fig. 4). Tissue reconstitution capacity in dediHep-derived hepatocytes was similar to that of hepatocytes freshly isolated from adult mouse livers (Fig. 3f). These data demonstrate that aggregation eliminates the mesenchymal phenotype in dediHeps, and facilitating re-differentiation into growth-arrested, functional hepatocytes.

dediHeps have an intestinal phenotype in monolayer culture

Given that dediHeps are undifferentiated cells with a hybrid E/M phenotype, we wondered whether they are able to differentiate into additional cell types. We discovered that more than 60% and 90% of dediHeps express the intestinal master transcription factor Cdx2 and its mRNA, respectively (Fig. 4a, b), normally expressed in intestinal epithelial cells but not in hepatocytes. Cdx2 was co-expressed with Alb in dediHeps (Fig. 4c) and gradually disappeared from dediHeps during re-differentiation into hepatocytes (Supplementary Fig. 5). In the development, bone morphogenetic protein (BMP) signaling is essential for mid/hindgut specification from definitive endoderm, inducing Cdx2 expression and the fate of intestine25,26. Also, ectopic expression of the BMP target gene inhibitor of differentiation (Id) 2 in the developing stomach induces Cdx2-positive intestinal epithelial cells in the gastric epithelia27. Thus, we examined whether BMP signaling is activated and critical for Cdx2 expression, in dediHeps. Sequential gene expression analyses during hepatocyte-to-dediHep conversion revealed that expression of BMP4, BMP7, and the BMP target genes Id1 and Id2 was increased in dediHeps (Fig. 4d). Moreover, inhibition of BMP signaling by Noggin significantly suppressed the expression level of Cdx2 in dediHeps (Fig. 4e). These data demonstrate that dediHeps express Cdx2 through activation of BMP signaling in monolayer culture.

Fig. 4: dediHeps express Cdx2 in response to BMP signaling.
figure 4

a Co-immunofluorescence staining (YFP with Cdx2) of dediHeps in monolayer culture. Graph at right depicts percentages of cells that were strongly or weakly positive for Cdx2 in YFP+ cells. b Cdx2 mRNA, but not the bacterial gene DapB mRNA (negative control), was detected in dediHeps by fluorescence in situ hybridization. E-cad protein was also detected in these dediHeps by immunofluorescence staining. Graph at right depicts percentages of Cdx2+ cells in E-cad+ cells. c Co-immunofluorescence staining (Alb with Cdx2) of dediHeps in monolayer culture. d Heatmap image from CEL-seq2 data showing the change in expression of the indicated genes during conversion of freshly isolated hepatocytes (FH) to dediHeps in monolayer culture 3 h, 1 day (d), 2 d, 4 d, 1 week (w), and 2 w after plating and at passage 10 (P10). e qPCR analysis of Cdx2 expression in dediHeps cultured with water or the inhibitor of BMP signaling Noggin for 4 days in monolayer culture. All data were normalized to the values for dediHeps cultured with water and are depicted as fold-changes. Statistical difference was determined by two-sided Student’s t test. DNA was stained with DAPI. Scale bars, 100 μm (a, c) and 20 μm (b). Data represent the mean ± SEM (n= 3 independent experiments). Source data are provided as a Source Data file.

dediHeps differentiate into intestinal epithelial cells that reconstitute colonic epithelia upon transplantation

The expression of Cdx2 in dediHeps suggested that they can differentiate into intestinal epithelial cells. To test this hypothesis, we embedded dediHeps in Matrigel 3D culture and treated them with epidermal growth factor (EGF), Noggin, and R-spondin1 (designated ENR) in the presence of Wnt3a and the Wnt agonist CHIR99021 (glycogen synthase kinase-3 inhibitor, designated WCENR), which induces fetal intestinal progenitor cells (FIPCs) to form spherical organoids (SOs)28. After 11 days of culture, dediHeps formed a small number of SOs, which could be propagated by serial passaging (Fig. 5a). The expression level of Cdx2 was increased and those of Alb and Vim were decreased after the formation and passages of SOs (Fig. 5b). These SOs were composed of actively proliferating, polarized intestinal epithelial cells that express E-cad basolaterally, Villin (also known as Vil1) apically, and nuclear Cdx2 and Sox9 (Fig. 5c and Supplementary Fig. 6a), similar to FIPC-derived SOs28,29. Also, dediHep-derived SOs were formed by cells expressing CK19 and Hnf4α but not Alb, indicating that these SOs were not biliary organoids that express CK19 but not Hnf4α and Alb (Supplementary Fig. 6b). Global gene expression analyses revealed that genes specifically expressed in dediHep-derived SOs or FIPC-derived SOs, compared with hepatocytes, are similar (Fig. 5d), and more than 97% of the detected genes were similarly expressed in both types of SOs (Fig. 5e). Moreover, addition of forskolin (FSK) with or without the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 to the culture media blocked or induced the swelling of dediHep-derived SOs, respectively (Supplementary Fig. 7). Thus, epithelial cells forming dediHep-derived SOs can functionally respond to FSK in a CFTR-dependent manner and increase the size of the SOs.

Fig. 5: dediHeps give rise to intestinal epithelial cells capable of reconstituting colonic epithelia upon transplantation.
figure 5

a Representative morphologies of dediHep-derived SOs at 11 days (d), passage 1 (P1), and P6 after initiation of 3D culture. Panel at lower-right is a representative micrograph of a hematoxylin and eosin (HE)-stained dediHep-derived SO. b qPCR analyses of expression of Alb, Cdx2, and Vim in dediHeps maintained in monolayer culture and SOs formed by dediHeps at 10 d and P4 after initiation of 3D culture. All data were normalized to the values for dediHeps in monolayer culture and are depicted as fold-changes. c Co-immunofluorescence staining (E-cad with Cdx2, Villin, Sox9, or Ki67) of dediHep-derived SOs. Graph at right depicts percentages of Cdx2+ cells, Villin+ cells, and Sox9+ cells in dediHep-derived SOs. d Volcano plots depicting differences in gene expression between hepatocytes and dediHep-derived SOs or FIPC-derived SOs. Each dot represents one gene. Blue and red dots represent down- and up-regulated genes, respectively, in dediHep-derived SOs or FIPC-derived SOs. Gray dots represent genes that are not significantly differentially expressed. e A Venn diagram shows direct comparisons of the global gene expression profiles between dediHep-derived SOs and FIPC-derived SOs. f Representative bright-field and fluorescence images of a recipient mouse colon 3 months after transplantation. Arrowheads indicate GFP+ donor-derived engraftments. g Co-immunofluorescence staining (GFP with Villin, Muc2, Cdx2, Klf5, EphB2, or Sox9) of recipient mouse colons 3 months after transplantation. h Transition of the body weights of mice injected with phosphate-buffered saline (PBS) (control) or dediHep-derived SOs for 7 days after second injection. All data represent the difference from the weights of the mice immediately before the second injection. DNA was stained with DAPI. Scale bars, 200 μm (a), 50 μm (a; HE staining), 20 μm (c, g), and 1 cm (f). Data represent the mean ± SEM (n= 3 independent experiments). Statistical difference was determined by one-way analysis of variance followed by Dunnett’s multiple comparison test (b), quasi-likelihood F-test (d), or two-sided Student’s t test (h). Source data are provided as a Source Data file.

In addition to our in vitro analyses, we sought to assess whether dediHep-derived intestinal epithalial cells could contribute to tissue reconstitution in vivo. It is known that FIPCs contribute to regeneration of adult colonic epithelium after transplantation into a chemically-induced colonic injury model29. Thus, we examined whether dediHep-derived intestinal epithelial cells behaved similarly. To this end, dediHep-derived SOs were transplanted into the colons of immunodeficient NOD/SCID/gamma (NSG) mice with dextran sulfate sodium (DSS)-induced acute colitis29,30. In mice transplanted with dediHep-derived SOs, donor cell clusters were macroscopically observed in colonic tissues 3 months after transplantation (Fig. 5f). Histology revealed that donor-derived cells were characterized as colonocytes expressing Cdx2 and Klf5 and gave rise to Villin+ absorptive cells, Muc2+ goblet cells, and EphB2+ and Sox9+ cells residing in the bottom of crypts (Fig. 5g). The body weights of mice transplanted with dediHep-derived SOs recovered faster than those of control mice (Fig. 5h). These data indicate that dediHep-derived SOs are capable of repopulating colonic epithelial tissues, similar to FIPC-derived SOs. Taken together, our data demonstrate that dediHeps can differentiate into not only hepatocytes, but also intestinal epithelial cells.

dediHeps give rise to intestinal stem cell (ISC)-like cells in response to forced expression of Cdx2

ISCs that reside at the bottoms of crypts in the small intestine continuously give rise to multilineage intestinal epithelial cells, including absorptive enterocytes, hormone-secreting enteroendocrine cells, mucin-secreting goblet cells, and antimicrobial protein-secreting Paneth cells, for replacing the epithelial cells lost by apoptosis at the villus tip31. ISCs form budding organoids (BOs) under 3D culture conditions, which consist of a lumen surrounded by a villus-like epithelial monolayer with budding crypt-like domains (CLDs)32. ISC-derived BOs are maintained in long-term culture by passaging with self-renewing cell divisions of ISCs, differentiation into multiple functional cells of the villi, and the release of apoptotic cells into the central lumen32. Although FIPCs do not form BOs, they are capable of developing into ISCs that form BOs by serial passaging in the presence of WCENR and subsequent exclusion of Wnt3a and CHIR99021 from the culture medium29. To examine whether dediHep-derived SOs could develop into BOs, dediHeps were subjected to 3D culture in the presence of WCENR, followed by removal of Wnt3a and CHIR99021 from the culture medium to promote their spontaneous maturation to BOs. However, unlike FIPC-derived SOs, dediHep-derived SOs did not form BOs during passaging, even after removal of Wnt3a and CHIR99021 from the culture medium (Fig. 6a). Our previous research has demonstrated that, in addition to endogenous Cdx2 expression, forced Cdx2 expression was required for the reprogramming of fibroblasts to FIPCs28. Thus, endogenous Cdx2 expression in dediHeps might be insufficient. To examine this possibility, dediHeps were infected with a retrovirus expressing Cdx2 and subjected to 3D culture (Fig. 6a−c). Forced expression of Cdx2 promotes the generation of SOs from dediHeps (Supplementary Fig. 8a). After serial passaging in the absence of Wnt3a and CHIR99021, these SOs developed into BOs that were morphologically indistinguishable from those derived from ISCs (Fig. 6a). Changing the additives in the medium from WCENR to ENR is necessary for the efficient formation of BOs even from dediHeps overexpressing Cdx2 (Supplementary Fig. 8b, c). dediHep-derived BOs could be maintained by repeated passaging in long-term 3D culture (Supplementary Fig. 8d).

Fig. 6: dediHeps give rise to ISC-like cells following forced expression of Cdx2.
figure 6

a Representative micrographs of 3D cultures of dediHeps transduced with GFP or Cdx2 (with GFP). Initially, dediHeps transduced with GFP or Cdx2 were embedded in Matrigel and cultured with WCENR. After passage 5, dediHep-derived SOs and BOs were cultured with ENR to promote the spontaneous transition of SOs to BOs. Crypts were consistently cultured with ENR. b qPCR analyses of Cdx2 expression by dediHeps transduced with GFP or Cdx2 (with GFP) 1 day (d) and 3 d after viral infection. All data were normalized to the value for dediHeps transduced with GFP at 1 d and are depicted as fold-changes. c Co-immunofluorescence staining (GFP with Cdx2) of dediHeps transduced with Cdx2 (with GFP) 1 day after viral infection. d Volcano plots depicting differences in gene expression between hepatocytes and dediHep-derived BOs or ISC-derived BOs. Each dot represents one gene. Blue and red dots represent down- and up-regulated genes, respectively, in dediHep-derived BOs or ISC-derived BOs. Gray dots represent genes that are not significantly differentially expressed. e A Venn diagram shows direct comparisons of the global gene expression profiles between dediHep-derived BOs and ISC-derived BOs. f Co-immunofluorescence staining (E-cad with Muc2, Villin, ChgA, Lyz, or CC3 and Sox9 with EphB2) of dediHep-derived BOs and ISC-derived BOs. Insets, representative ChgA+ E-cad+ cells. g qPCR analyses of expression of Cdx2 and Vil1 and marker genes for differentiated intestinal epithelial cells (Lyz1, Chga, and Muc2) and ISCs (Lgr5 and Ascl2) in hepatocytes freshly isolated from adult mouse livers, dediHeps maintained in monolayer culture, SOs formed by dediHeps transduced with GFP, BOs formed by dediHeps transduced with Cdx2 (with GFP), and ISC-derived BOs. All data were normalized to the values for ISC-derived BOs and are depicted as fold-changes. DNA was stained with DAPI. Scale bars, 200 μm (a) and 50 μm (c, f). Data represent means ± SEM (n = 3 independent experiments). Statistical difference was determined by two-sided Student’s t test (b), quasi-likelihood F-test (d), or one-way analysis of variance followed by Dunnett’s multiple comparison test (g). Source data are provided as a Source Data file.

Global gene expression analyses revealed that genes specifically expressed in dediHep-derived BOs or ISC-derived BOs, compared with hepatocytes, are similar (Fig. 6d), with more than 97% of the detected genes being expressed similarly in both types of BOs (Fig. 6e). Immunofluorescence analyses revealed that the cells comprising dediHep-derived BOs consistently expressed basolateral E-cad and apical Villin, indicating that they were typical intestinal epithelial cells, mainly enterocytes, that maintain distinctive apicobasal cell polarity (Fig. 6f). These BOs also contained Paneth cells, enteroendocrine cells, and goblet cells, which were respectively characterized by the expression of Lysozyme (Lyz; also known as Lyz1), Chromogranin A (ChgA), and Muc2 (Fig. 6f). Moreover, the expression of ISC-enriched Wnt target proteins, such as Sox9 and EphB2, in BOs were restricted to the CLDs, and cleaved caspase 3 (CC3)-positive apoptotic cells were released into their central lumens (Fig. 6f). These characteristics closely resemble those of ISC-derived BOs (Fig. 6f). Quantitative polymerase chain reaction (qPCR) analyses also revealed that the expression levels of differentiated intestinal epithelial cell markers, such as Lyz1, Chga, and Muc2, and those of dediHep markers, such as Alb and Vim, significantly increased and decreased, respectively, in BOs derived from exogenous Cdx2-expressing dediHeps (Fig. 6g and Supplementary Fig. 9). These data demonstrate that forced Cdx2 expression promotes the generation of ISC-like cells from dediHeps, which form BOs and give rise to multilineage intestinal epithelial cells.

dediHeps are partially similar to chemically induced liver progenitors (CLiPs), but differs in many aspects

Small molecule compounds, such as the Rho-associated kinase inhibitor Y-27632, the TGF-β type I receptor inhibitor A-83-01, and CHIR99021, allowed hepatocyte dedifferentiation in serm-free monolayer culture, and the resultant cells were named CLiPs12. To compare dediHeps with CLiPs, we cultured adult mouse hepatocytes in accordance with the published protocol and induced hepatocyte dedifferentiation into CLiPs. CLiPs required a couple of months to proliferate sufficiently, while dediHeps needed only a couple of weeks. In fact, the proliferative capacity of CLiPs was significantly lower than that of dediHeps (Supplementary Fig. 10a). CLiPs were morphologically identified as epithelial cells and expressed Alb, CK19, E-cad, and Vim, similar to dediHeps (Fig. 7a), suggesting that CLiPs also have a hybrid E/M phenotype. Global gene expression analyses revealed that expression levels of Cdh1, Cldn3, Ocln, Vim, Prom1, Hnf4α, and Hnf1α were similar, but those of S100a7a, Lamb1, Mki67, and Krt19 were lower, in CLiPs relative to their expression in dediHeps (Fig. 7b). Meanwhile, several genes encoding liver enzymes were highly expressed, and the expression level of Cdx2 was significantly lower, in CLiPs compared to their expression in dediHeps (Fig. 7b). Dedifferentiation of human hepatocytes into CLiPs14 also activates the expression of the mesenchymal cell markers VIM and LAMB1, in addition to PROM1, MKI67, and KRT19 (Supplementary Fig. 10b). Thus, CLiPs partially resemble dediHeps with a hybrid E/M phenotype but have more characteristics as hepatocytes than dediHeps.

Fig. 7: dediHeps are different from CLiPs and give rise to both hepatic and intestinal lineage cells.
figure 7

a Representative morphologies and fluorescence micrographs of CLiPs expressing Alb, CK19, E-cad, and Vim. DNA was stained with DAPI. Scale bars, 100 μm. b Volcano plots depicting differences in gene expression between dediHeps and CLiPs. Each dot represents one gene. Blue and red dots represent genes whose expression are lower and higher in CLiPs than dediHeps, respectively. Gray dots represent genes that are not significantly differentially expressed. Statistical difference was determined by quasi-likelihood F-test. c PCA was performed using CEL-seq2 data for freshly isolated hepatocytes (FH), hepatocytes (Hep) in monolayer culture 3 h, 1 day (d), 2 d, 4 d, 1 week (w), and 2 w after plating, dediHeps in monolayer culture, dediHep aggregates, dediHep-derived SOs, FIPC-derived SOs, dediHep-derived BOs, ISC-derived BOs, and CLiPs.

To assess the relationship among various types of cells analyzed in this study, we investigated the global gene expression profiles in these cells and performed principal component analysis (PCA) (Fig. 7c). PCA revealed that the gene expression signatures of hepatocytes changed widely after 1 day in monolayer culture and were stepwisely close to those of dediHeps as the culture progresses. The gene expression signatures of CLiPs were most similar to those of hepatocytes cultured for 1 week. Upon cell-aggregate formation, the gene expression signatures of dediHeps approached those of hepatocytes cultured for a few days in monolayer culture. The formation of SOs in Matrigel 3D culture moved the gene expression signatures of dediHeps away from those of hepatic lineage cells and bring them closer to those of FIPC-derived SOs. In addition, dediHep-derived BOs and ISC-derived BOs occupy a similar dimensional space, indicating a close resemblance between the gene expression signatures of two different types of BOs.

Hippo inactivation in hepatocytes is fundamental for their conversion to dediHeps

We next sought to identify the mechanism through which hepatocytes convert to dediHeps. Genes associated with the Hippo signaling pathway were up-regulated in dediHeps and down-regulated in dediHep-derived, re-differentiated hepatocytes (Figs. 2d and 3d). Also, hepatocyte dedifferentiation is induced in vitro and in vivo by activation of Yap, a central effector molecule of the Hippo signaling pathway8,10. Thus, it is suggested that Yap activation is important for the conversion of hepatocytes to dediHeps. Immunofluorescence analyses revealed that Yap protein accumulated in the nucleus of hepatocytes immediately after their dissociation from the liver, which was maintained throughout the period of hepatocyte culture and in the resultant dediHeps (Fig. 8a, b). The expression of the Yap target genes cysteine rich-61 (Cyr61) and connective tissue growth factor (Ctgf) was significantly increased during culture and maintained in dediHeps (Fig. 8c). Gene set enrichment analysis (GSEA) revealed that Yap-associated genes were up-regulated in dediHeps compared with hepatocytes (Fig. 8d). Meanwhile, Yap immediately disappeared from the nucleus of dediHeps as a result of cell-aggregate formation that induced re-differentiation of dediHeps into growth-arrested functional hepatocytes under 3D culture conditions (Fig. 8e and Supplementary Fig. 11). To synchronize with this, expression levels of Cyr61 and Ctgf also decreased (Fig. 8f).

Fig. 8: Conversion of hepatocytes to dediHeps requires Yap activation.
figure 8

a Co-immunofluorescence staining (Hnf4α with Yap) of adult mouse liver tissues, hepatocytes freshly isolated from adult mouse livers (FH), and hepatocytes in monolayer culture 3 h, 1 day (d), 2 d, 4 d, and 1 week (w) after plating and at passage 4 (P4). Insets, subcellular localization of Yap protein. b Percentages of Hnf4α+ cells positive for nuclear Yap. c qPCR analyses of Cyr61 and Ctgf expression in liver tissues and the indicated cells. All data were normalized to the values for freshly isolated hepatocytes (FH)−1 and are depicted as fold-changes. d GSEA of whole-transcriptome data from dediHeps and freshly isolated hepatocytes using a set of YAP-associated genes. e Immunofluorescence staining of Yap was conducted for dediHeps in monolayer culture and dediHep aggregates at the indicated days (d) after initiation of 3D culture. The graph shows the percentages of cells marked with nuclear Yap in monolayer and 3D cultures of dediHeps. f qPCR analyses of Cyr61 and Ctgf expression in dediHeps maintained in monolayer culture and dediHep aggregates 1 d, 3 d, and 5 d after initiation of 3D culture. All data were normalized to the values for dediHeps in monolayer culture and are depicted as fold-changes. g Representative micrographs of hepatocyte cultures treated with DMSO or verteporfin (0.1, 0.2, and 0.3 μM) 1 d, 4 d, 1 w, and 2 w after plating. h Growth of hepatocyte-derived cells cultured with DMSO or verteporfin (0.1, 0.2, and 0.3 μM). i Heatmap image from CEL-seq2 data showing that the expression levels of the indicated genes differ between DMSO- and verteporfin (0.3 μM)-treated dediHeps. DNA was stained with DAPI. Scale bars, 50 μm (a) and 200 μm (g). Data represent means ± SEM (b, e, f, and h) or SD (c) (n = 3 independent experiments). Statistical difference was determined by one-way analysis of variance followed by Dunnett’s multiple comparison test (b, c, e, f, and h) or permutation test (d). Source data are provided as a Source Data file.

To examine the importance of Yap activation in hepatocytes, we treated them with verteporfin, an inhibitor of Yap transcriptional activity. This inactivation blocked the conversion of hepatocytes to dediHeps in a concentration-dependent manner in verteporfin (Fig. 8g, h) and led to higher expression of several genes encoding liver enzymes and lower expression of Vim and Prom1 than hepatocytes cultured without verteporfin (Fig. 8i and Supplementary Fig. 12). Since Yap is activated in FIPC-derived SOs and its inhibition impairs SO formation33, we examined the role of Yap activation in dediHep-derived SOs. Our data demonstrated that dediHep-derived SOs were composed of cells marked with nuclear Yap, and their formation was nearly completely blocked in culture with verteporfin (Supplementary Fig. 13). Thus, Yap activation is critical for the induction of the hybrid E/M phenotype in cultured hepatocytes, their conversion to dediHeps, and differentiation of dediHeps into intestinal epithelial cells.

Hepatocyte-to-dediHep conversion occurs independently of TGF-β signaling, and Vim is required for the maintenance of dediHeps in culture

Our present data demonstrate that primary hepatocytes acquire a hybrid E/M phenotype in the early stages of culture, which was maintained during and after the conversion of hepatocytes into dediHeps. It is well-known that TGF-β signaling plays an important role in EMT34. Thus, to determine whether TGF-β signaling was involved in the conversion of hepatocytes into dediHeps, we isolated hepatocytes from the livers of wild-type mice and Alb-Cre;Tgfbr2fl/fl mice (deficient in the liver-specific TGF-β receptor 2), then cultured them in the presence or absence of TGF-β. We found that TGF-β signaling was toxic to hepatocytes and unnecessary for their conversion to dediHeps (Fig. 9a, b). TGF-β signaling induced EMT by completely suppressing E-cad expression, while Vim expression was induced in cultured hepatocytes both with and without TGF-β activation (Fig. 9c). These results indicate that the mesenchymal phenotype appearing in cultured hepatocytes is induced in a TGF-β-independent manner, and the hybrid E/M cell state is required for the generation of dediHeps.

Fig. 9: Roles of TGF-β signaling and Vim in the induction and maintenance of dediHeps in culture.
figure 9

ac Hepatocytes isolated from the livers of adult wild-type and Alb-Cre;Tgfbr2fl/fl mice cultured with citric acid or TGF-β. a Representative micrographs of hepatocyte cultures 1 day (d), 4 d, 1 week (w), 2 w, and 3 w after plating. b Growth of hepatocyte-derived cells. c Co-immunofluorescence staining (E-cad with Vim) of hepatocyte-derived cells at 2 w in culture. DNA was stained with DAPI. d qPCR analysis of Vim expression in dediHeps expressing GFP with a control shRNA against lacZ (shLacZ), shVim986, or shVim1183. All data were normalized to the values for dediHeps expressing shLacZ (shLacZ-1) and are depicted as fold-changes. e Representative morphologies and fluorescence micrographs of GFP+ dediHeps expressing shLacZ, shVim986, or shVim1183. f Percentages of GFP+/sh+ (shLacZ, shVim986, or shVim1183) dediHeps that express phospho-histone H3 (pHH3). g qPCR analyses of Alb and Cdx2 expression in dediHeps expressing shLacZ or shVim1183. All data were normalized to the values for dediHeps expressing shLacZ and are depicted as fold-changes. h Representative morphologies and the number (right graph) of SOs formed by dediHeps expressing shLacZ or shVim1183 at 11 d after initiation of 3D culture. Scale bars, 200 μm (a, h), 50 μm (c), and 100 μm (e). Data represent means ± SEM (b, f, g, and h) or SD (d) (n = 3 independent experiments). Statistical difference was determined by two-sided Student’s t test (b, g, and h) or one-way analysis of variance followed by Dunnett’s multiple comparison test (d, f). Source data are provided as a Source Data file.

One of the major mesenchymal features of dediHeps is Vim expression. Thus, we next investigated its role. To this end, we used two kinds of short hairpin RNA (shRNA) targeting Vim, designated shVim986 and shVim1183, to reduce Vim expression (Fig. 9d). The dediHeps in which Vim had been knocked down were morphologically different from, and proliferated much slower than, dediHeps expressing a control shRNA (Fig. 9e, f). Moreover, by knocking down Vim expression, the expression levels of Alb and Cdx2 were decreased and increased, respectively, in dediHeps (Fig. 9g), and the number of SOs formed from dediHeps was increased (Fig. 9h). Thus, Vim is essential for the maintenance of dediHeps in culture by inducing their proliferation and interfering intestinal differentiation.