Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells Free

Abstract

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells  2019;37:306–317

Significance Statement

Using human induced pluripotent stem cells, it is discovered that a protein kinase c-Src is important for endoderm formation and that a transient alteration of its activity in the early stage of liver development results in abnormal fibrotic-biliary cells resembling those observed in patients with biliary fibrosis. As it is not feasible to study early endoderm formation in human fetus, the study provides new insights on endoderm development in human and a basis for establishing human-specific experimental systems for modeling abnormal liver development that may be potentially associated with hepatobiliary cirrhosis.

Cell Fate Diversion Resulting from Transient Src Inhibition During Early Endodermal Commitment of Human Pluripotent Stem Cells

Inhibition of Src kinase activity during endodermal commitment of human induced pluripotent stem cells results in significantly reduced endodermal and hepatic specification in a dose-dependent manner and a cell fate change to cells resembling abnormal fibrogenic cholangiocyte-like cells.

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Introduction

The molecular and cellular changes involving endodermal specification and hepatogenesis in humans are not clearly defined because of a lack of human-relevant models. The majority of developmental cues discovered have come from organogenesis/developmental studies that have heavily relied on nonhuman model systems [1-6]. The potential to reprogram differentiated human cells into a pluripotent state has opened up new avenues to study varied cellular fate commitment during tissue-specific differentiation process of human induced pluripotent stem cells (iPSCs) [7-21]. Differentiation protocols for inducing hepatobiliary tissues from human iPSCs are multiphasic, mimicking the multistage development of liver in vivo, and involve the definitive endodermal specification, hepatic commitment, and further maturation stages into either hepatocytes or biliary tissues [7, 8, 11, 14, 15, 18]. Nodal signaling, initiated by activin A, has a critically important role in the induction of endoderm [22, 23]; thus, effective protocols for endoderm differentiation of human iPSCs have used activin A [18, 22, 24-27]. In addition, there are fundamental differences in mouse and human iPSCs in terms of maintaining pluripotency and differentiating them into hepatic cells [17, 28]. Human iPSCs may therefore be the most human-relevant tool available to evaluate the essential signaling elements required for the development of hepatobiliary tissues.

The purpose of this study was to elucidate the key underlying molecular mediators regulating human liver development. By using a panel of kinase inhibitors specifically targeting a majority of known signaling pathways, we identified a previously unappreciated role of c-Src in the endoderm commitment and hepatobiliary differentiation of human iPSCs in this study. Src family kinases (SFKs) comprise 11 Src-related kinases in the human genome [29], which in conjunction with various growth factor receptors play diverse roles in cellular processes such as survival, proliferation, differentiation, and cell adhesion [30-33]. Of these, eight have been studied extensively in mammalian cells (Blk, Fgr, Fyn, Lck, Lyn, Hck, c-Src, and c-Yes) [30, 31]. Of the 11 members of SFKs, Fyn, Yes, Src, Lck, Lyn, and Hck are expressed in human embryonic stem cells (ESCs) [34], whereas murine ESCs express seven members of the SFKs, of which Src, Hck, and Fyn are constitutively expressed in self-renewing ESCs [31]. The transcriptional expression levels of Yes, Hck, and Lyn remain constant in self-renewing human ESCs. Lck level reduces drastically on embryoid body (EB) differentiation, whereas a modest increase in expression of Src and Fyn is seen on EB differentiation of human ESCs [34]. Lck and Yes activity may thus have a role in self-renewal, whereas Src and Fyn may power human ESC differentiation. It has also been shown that, Src is highly active following retinoic acid-induced human ESC differentiation [35]. These previous studies together support the importance of Src in differentiation and development of mammalian tissues. Here, we propose that c-Src activity is critical for endodermal commitment of human iPSCs and present its fate-governing role in human hepatobiliary differentiation and potential implication in biliary fibrosis pathogenesis [36-38].

Methods

Endodermal Commitment and Hepatic Differentiation of Human iPSCs

All human iPSCs used in this study were previously generated in our laboratory [8, 11, 18-20]. This study was conducted in accordance with Johns Hopkins https://www.hopkinsmedicine.org/research/resources/offices-policies/iscro/index.htmlInstitutional Stem Cell Research Oversight Committee regulations and following a protocol approved by the Johns Hopkins Institutional Review Board. The human iPSCs were cultured in feeder-free condition on Matrigel (BD Biosciences), using mTeSR (Stem Cell Technologies) medium as previously described [7, 8, 11, 18]. The medium was replaced every day until the cells reach the desired confluence for passaging or differentiation. Once the human iPSCs reached a 40%–60% confluence status, endodermal commitment and hepatic differentiation was induced as previously described [7, 8, 11, 18], using Roswell Park Memorial Institute (RPMI) medium supplemented with 30–100 ng/ml activin A and 1 μM CHIR-99021 on day 0. This medium was replaced with RPMI medium supplemented with activin A and B27 supplement from day 1 to day 5 to generate definitive endoderm (DE) cells. Hepatic commitment was induced by culturing endoderm cells in RPMI medium supplemented with cytokines hepatocyte growth factor (HGF) and fibroblast growth factor 4 (FGF4), 10 ng/ml each, and B27 supplement (hepatic progenitor [HP] medium) for further 4–5 days to obtain HP cells. Hepatic maturation of HP cells to mature hepatocyte stage was stimulated by culturing HP cells for 10–15 days in hepatocyte culture medium supplemented with the cytokines HGF, FGF4, oncostatin M at a concentration of 10 ng/ml, and 0.1 μM dexamethasone. Eighty inhibitors in the Tocriscreen Kinase Inhibitor Toolbox (Tocris #3514) were initially screened individually by testing at a 2.5 μM dosage. Src inhibition was achieved by incorporation of Src inhibitor 1-naphthyl PP1 (NPP1; Tocris) into culture media for 3 days at a final concentration of 0.25–1 μM at different stages. For verteporfin (VP; Sigma) treatment, NPP1 was incorporated during initial 3 days (days 0–2) of endodermal commitment, followed by addition of VP from days 5 to 7, and cells were evaluated at HP stage (days 8–10).

Mesodermal and Ectodermal Commitment of Human iPSCs

Human iPSCs were allowed to become 60%–70% confluent before mesodermal or ectodermal commitment was induced. Mesodermal differentiation was induced by culturing in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with GlutaMax, B27 supplement, 1%PenStep, 3 μM CHIR99021, and 1 μM 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid, Arotinoid acid (TTNPB) for 2 days, followed by 4-day culture in same medium without CHIR99021 [39]. Ectodermal differentiation was induced by culturing for 6 days in DMEM/F12 medium supplemented with 4% knockout serum replacement (KOSR), 1% non-essential amino acids (NEAA), 1% PS, 5 μM SB431542, and 100 nM LDN-193189 [40].

Determination of Transcriptional Expression of Markers by RNA Extraction and Quantitative-Polymerase Chain Reaction

TRIZOL reagent (Invitrogen) was used to extract total RNA. A High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for reverse transcription from mRNA to cDNA. Twenty nanograms of resulting cDNA was used as a template for real-time polymerase chain reaction (PCR). Quantitative PCR was carried out with the StepOnePlus Real-Time PCR System (Applied Biosystems), using TaqMan primer-probes: OCT4 (Hs03005111_g1), NANOG (Hs04260366_g1), Src (Hs01082246_m1), SOX17 (Hs00751752_s1), FOXA2 (Hs00232764_m1), CXCR4 (Hs00607978_s1), BRACHYURY (Hs00610080_m1) Mesoderm Posterior BHLH Transcription Factor 1 (MESP1) (Hs01001283_g1), Paired Box 6 (PAX6) (Hs00240871_m1), Hepatocyte Nuclear Factor 4 Alpha (HNF4A) (Hs01023298_m1), Hepatocyte Nuclear Factor 4 Alpha (AFP) (Hs00173490_m1), Epithelial Cell Adhesion Molecule (EpCAM) (Hs00901885_m1), CCAAT Enhancer Binding Protein Alpha (CEBPA) (Hs00269972_s1), CCAAT Enhancer Binding Protein Alpha (CEBPB) (Hs00942496_s1), CK7 (Hs00559840_m1), CK19 (Hs00761767_s1), SOX9 (Hs01001343_g1), Neural Cell Adhesion Molecule 1 (NCAM1) (Hs00941830_m1), Aquaporin 1 (AQP1) (Hs01028916_m1), GPBAR1 (Hs01937849_s1), LOXL2 (Hs00158757_m1), COL1A1 (Hs00164004_m1), SMA (Hs00426835_g1), MMP2 (Hs01548727_m1), TIMP1 (Hs01092512_g1), TIMP2 (Hs00234278_m1), LOXL2 (Hs00158757_m1), PDGFC (Hs00211916_m1), Vimentin (VIM) (Hs00958111_m1), YAP1 (Hs00902712_g1), SNAI2 (Hs00161904_m1), CDH1 (Hs01023895_m1), IL-6 (Hs00985639_m1), Tumor Necrosis Factor Alpha (TNFα) (Hs00174128_m1), TGF (Hs00998133_m1), and 18S rRNA (Hs03003631_g1) in a total volume of 20 μl. 18S was used as a housekeeping gene for normalization of gene expression results using StepOnePlus Real-Time PCR System (Applied Biosystems).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde (Sigma) for 20 minutes at room temperature. Cells were incubated with appropriate primary antibodies at 4°C overnight and then washed twice and incubated with Alexa Flour 555 or 488 secondary antibodies (Invitrogen) in phosphate-buffered saline (PBS) at room temperature for 45 minutes. Finally, cells were counterstained with 4′,6-diamidino-2-phenylindole. Primary antibodies against octamer-binding transcription factor 4 (OCT4; 1:200, Millipore), TRA1-60 (1:100, Millipore), NANOG (1:100, BD Pharmingen), SOX17 (1:200, R&D Systems), chemokine receptor type 4 (CXCR4; 1:100, eBioscience), AFP (1:200, Dako), CK7 (1:200, Cell Marque), CK7 (1:200, Millipore), smooth muscle actin (SMA; 1:200, Sigma), COL1A1 (1:100, Calbiochem), and yes-associated protein 1 (YAP1; 1:100, Sigma) were diluted in PBS with 0.3% bovine serum albumin and 0.1% Triton X-100. Images were taken using the motorized Nikon Ti-E microscope and NIS-Elements software. Quantification of protein expression and cell counting were done using ImageJ software (http://imagej.nih.gov/ij/).

Senescence Beta-Galactosidase Staining

The control and cholangiocyte-like cells arising from Src inhibition during endodermal commitment of human iPSCs were fixed with 4% paraformaldehyde (Sigma) for 20 minutes at room temperature. Fixed cells were stained as per manufacturer's protocol (Cell Signaling Technology #9860). Images were taken using the motorized Nikon Ti-E microscope and NIS-Elements software.

Flow Cytometry

HP stage cells were fixed and incubated with CK7 (1:200, Millipore) antibody for 30 minutes at room temperature. Cells were washed twice and then resuspended with corresponding AlexaFlour antibody in PBS. Flow cytometric analysis was performed using the Guava easyCyte Flow Cytometer (Millipore).

Enzyme-Linked Immunosorbent Assay

Total cellular protein was extracted and sandwich enzyme-linked immunosorbent assay (ELISA) for phosphorylated Src (Tyr416; Cell Signaling Technology #7953C) was performed by following manufacturer's protocol. ELISA for interleukin-6 (IL-6; ThermoFisher #EH2IL6) and TNFα (ThermoFisher Scientific #KHC3011) was performed as per manufacturer's protocol using cell culture supernatents.

Western Blot

Cells were washed with PBS and lysed with 200 μl prepared cold RIPA buffer with protease and phosphatase inhibitors per 10⁷ cells (50 μl 100x protease inhibitors [Sigma P8340], 50 μl 100x 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) 3 [Ser-Thr phosphatase inhibitor {Sigma P5726}], 50 μl 100x PPI 2 [Tyr- phosphatase inhibitor {Sigma P5726}]). Cell lysates were clarified with centrifugation, and equal amounts of total protein was mixed with loading buffer, heated, and separated on SDS polyacrylamide gels. Proteins were then transferred onto Polyvinylidene difluoride (PVDF) membranes and probed with active Src (phospho Y418) and actin (Santa Cruz; sc-47,778) antibodies. Both primary antibodies and corresponding secondary antibodies were used at 1:1000 dilution. All Western blot film images were cropped to show adequate bandwidth above and below relevant bands.

Statistics

Data are expressed as mean ± SEM. For pairwise comparisons, a Student's t test was used. For all tests conducted, p < .05 was considered significant.

Results

Src Kinase Activity Is Essential for Endodermal Commitment of Human iPSCs

To elucidate the underlying cellular mediators regulating endodermal and hepatic commitment of human iPSCs (Supporting Information Fig. S1A), we screened a panel of small molecule inhibitors targeting specific tyrosine kinases (Tocriscreen Kinase Inhibitor Toolbox #3514; Supporting Information Fig. S1B; Table S1) in each stage of multistage hepatic differentiation of human iPSCs. Treatment with a majority of these kinase inhibitors did not show any clear difference from untreated control cells at any stage in the hepatocyte differentiation process, except three inhibitors (PP1, PP2, and NPP1; Supporting Information Table S1; Fig. S1C, S1D). These three different inhibitors, all targeting c-Src, showed the same result in endoderm commitment stage. While the control cells differentiated to DE cells, those grown in the presence of Src kinase inhibitors PP1, PP2, and NPP1 grew morphologically similar to undifferentiated colonies (Supporting Information Fig. S1C) and maintained expression of NANOG, a pluripotency marker (Supporting Information Fig. S1D).

Transcriptional assessment of the cells immediately after the treatment with a more specific Src inhibitor, NPP1 (Fig. 1A; Supporting Information Fig. S2A), revealed that they retain higher transcriptional expression levels of pluripotency marker OCT4 than day 3 DE, whereas the expression of DE markers SOX17, FOXA2, and CXCR4 was significantly reduced in a dose-dependent manner (Fig. 1B). Furthermore, these NPP1-treated cells retained high protein expression of pluripotency markers OCT4, TRA-1-60, and NANOG (Fig. 1C), whereas the expression of endodermal marker proteins SOX17 and CXCR4 was much reduced (Fig. 1D). Quantification of protein expression of these markers was done using ImageJ software (http://imagej.nih.gov/ij/; Fig. 1E).

We investigated whether Src expression was induced during directed endodermal commitment of iPSCs (Supporting Information Fig. S2). Both Src expression and Src phosphorylation at tyrosine 416, indicative of activation of c-Src, were increased in DE cells, but these were reduced in the NPP1-treated cells. NPP1 treatment, however, did not adversely affect or inhibit mesodermal or ectodermal commitment (Supporting Information Fig. S3A, S3B), whereas it reduced endodermal commitment (Fig. 1; Supporting Information Figs. S2, S3), indicating that Src is not indispensable for mesodermal or ectodermal commitment as it is for endodermal commitment. These results together indicate the key role that Src plays in endodermal commitment of human iPSCs.

Inhibition of Src Kinase Activity During Endodermal Differentiation of Human iPSCs Promotes an Early Biliary Fate

These cells at day 3 of endodermal differentiation with NPP1 treatment (Fig. 2A) showed increased transcriptional levels of genetic markers associated with early biliary development CEBPB [41], SOX9 [15], and expressed significantly increased levels of cholangiocyte marker CK7 [16] in a dose-dependent manner (Fig. 2B). In addition, the NPP1-treated cells showed increased protein expression of CK7 at day 3 of DE stage (Fig. 2C). On prolonged culture of transiently NPP1-treated cells (at day 5; Fig. 2D), the treated cells showed further enhanced expression of early biliary differentiation markers CEBPB, SOX9, and a more dramatic increase of mature cholangiocyte markers CK7 as well as CK19 [16] (Fig. 2E, 2F).

Inhibition of Src Kinase Activity During Endodermal Commitment of Human iPSCs Alters Subsequent Hepatocytic Commitment, and Gives Rise to Cells Expressing Cholangiocyte Markers in a Prolonged Hepatic Differentiation Culture Condition

To evaluate the cell fate change observed after transient inhibition of Src during endodermal commitment, after the 3 days treatment with Src inhibitor, we continued the culture of these cells in the directed hepatic differentiation environment, as per our pre-established hepatic differentiation protocol (Fig. 3A) [7, 8, 11, 18]. We compared the cells obtained at HP stage (differentiation days 8–10) from transient NPP1-treated cells during endodermal commitment, with untreated control HP cells at the same stage arising from control DE cells (Fig. 3A). The cells arising at HP stage from NPP1-treated cells during endodermal commitment showed more elongated morphology in comparison to normal HP cells (Supporting Information Fig. S4A; Fig. 3F, 3H). Transcriptional evaluation revealed that NPP1-treated cells showed decreased expression of markers associated with early hepatocytic commitment from HP including CEBPA, HNF4A, AFP, and a marker for both HP and biliary cells, EpCAM [15] (Fig. 3B). Protein expression of HP marker AFP (Fig. 3D) and EpCAM (Fig. 3E) was reduced in these NPP1-treated cells. Src expression during endoderm commitment stage is thus vital for the subsequent commitment toward the HP stage.

Intriguingly, these NPP1-treated cells further cultured in hepatic specification environment instead showed increased expression of early biliary-commitment-associated genes such as CEBPB, SOX9, and NCAM1 (Fig. 3B). Furthermore, expression of mature cholangiocyte markers CK7, AQP1, CK19, and GPBAR1 [16] (Fig. 3B) also increased significantly. Flow cytometry and fluorescence-activated cell sorting analysis and immunofluorescence imaging also revealed a remarkable increase in the protein expression of mature cholangiocyte markers, CK7 (Fig. 3C, 3F), acetylated tubulin [16] (Fig. 3G; Supporting Information Fig. S5C), and CK19 (Fig. 3H) in NPP1-treated cells. Quantification of protein expression of these markers was done using ImageJ software (Fig. 3J).

Transient Src inhibition during endodermal commitment thus clearly diverts the cellular fate from normal hepatocytic differentiation to a more cholangiocyte-like cell type via a primitive ductal progenitor (DP) stage.

The Cholangiocyte-Like Cells Emerging from Prolonged Hepatic Culture after Transient Src Inhibition During Endodermal Commitment Showed Increased Expression of Fibrosis, YAP, and Inflammation Markers

These cholangiocyte-marker expressing cells at days 8–10 (Fig. 3) unexpectedly showed increased expression of markers associated with hepatic fibrosis such as collagen type 1 (COL1), SMA, Matrix Metallopeptidase 2 (MMP2), TIMP Metallopeptidase Inhibitor 1 (TIMP1), TIMP Metallopeptidase Inhibitor 2 (TIMP2), Lysyl Oxidase Like 2 (LOXL2), Platelet Derived Growth Factor C (PDGFC), VIM, and Transforming Growth Factor Beta 1 (TGFβ) at equivalent levels to those of hepatic stellate cells (LX2) (Fig. 4A). Increased expression of markers associated with inflammation such as IL-6 [42, 43] and TNFα (Fig. 4E) was also observed in the cholangiocyte-like cells resulting from Src inhibition during endodermal commitment. Cells coexpressing CK7 and COL1 increased strikingly (Fig. 4B). Concurrently, we observed an increased expression of SMA and increased number of cells coexpressing SMA and CK7 (Fig. 4C). Furthermore, we observed an increased transcriptional (Fig. 4A) and protein expression (Fig. 4D) of YAP, which has been reported as a marker associated with bile duct proliferation and biliary fibrosis [37, 38], with a dramatic increase in the cells coexpressing CK7 and YAP. Furthermore, the protein expression of inflammatory markers IL-6 and TNFα (Fig. 4F) increased significantly, with increased secretion of these inflammatory cytokines by the NPP1-treated cells. These cholangiocyte-like cells arising from transient Src inhibition during endodermal commitment thus showed fibrotic inflammatory phenotypes.

To further study the development of these aberrant cholangiocyte-like cells, we analyzed the kinetics of expression of both biliary markers and fibrosis markers post Src inhibition during endodermal commitment (Fig. 5). Immunofluorescence staining further demonstrated the increased protein expression of CK7 (Fig. 5A), COL1 (Fig. 5A, 5B), and YAP1 (Fig. 5B). CK7 positive cells start appearing as early as day 5 and increase dramatically in number on further culture (Fig. 5A). These CK7 positive cells start acquiring a fibrotic potential around day 5 as observed by the expression of COL1 (Fig. 5A, 5B), and the expression as well as percentage of cells coexpressing CK7 and COL1 increases (Fig. 5A) as differentiation progresses. We further observed that YAP expression remains nuclear and robustly active until day 7, after which we observed cytoplasmic YAP expression (Fig. 5B). With continued culture, the expression and the number of cells coexpressing YAP1 and COL1 again strikingly increased (Fig. 5B). Furthermore, many of these CK7+ cholangiocyte-like cells were proliferating [44] from day 5, and the cell size increases along with proliferation (Fig. 5C). Transcriptional analysis of CK7, CK19, YAP1, COL1, and SMA revealed increased expression of these biliary and fibrosis markers, as the expression of a pluripotency marker OCT4 decreases in cells treated with NPP1 during endodermal commitment (Fig. 5D) as compared to stage-specific control cells. One of the epithelial-to-mesenchymal transition (EMT) markers, Snail Family Transcriptional Repressor 2 (SNAI2), was increased, although many other EMT markers including TWIST1/2, SNAI1, ESRP1/2, and ZEB1 did not increase in these cells (data not shown). In addition, an epithelial marker E-cadherin (also known as CDH1) did not decrease in these cholangiocyte like cells (Fig. 5D).

In human liver diseases, cholangiocytes presenting with a fibrotic and inflammatory phenotype are also associated with increased expression of senescence phenotypes [45-47]. Hence, we analyzed the expression of senescence phenotypes including senescence-associated β-galactosidase (SA-β-GAL) and cell cycle regulators such as p16^INK4a, P21, and P53 in the proinflammatory fibrotic cholangiocyte-like cells arising from Src inhibition during endodermal commitment (Supporting Information Fig. S6). The expression level of P16 was more dramatically increased at the day 8 cells, whereas SA-β-GAL and other cellular senescence markers (P21 and P53) showed marginal increase. In addition, protein secretion of IL-6, an important cytokine in human hepatobiliary pathology such as biliary atresia (BA), primary sclerosing cholangitis (PSC), and cholangiocarcinoma [42, 43], was also more significantly increased in the day 8 cells (Supporting Information Fig. S7). Together, these results suggest that the senescence phenotype might be at least partially acquired by the proinflammatory, fibrotic cholangiocytes arising from the transient Src inhibition during endodermal commitment of iPSCs in hepatic culture conditions.

Treatment with VP, a YAP inhibitor [37, 48, 49], significantly decreased the transcriptional expression of markers associated with fibrosis such as YAP1, COL1, SMA, TIMP1, MMP2, and LOXL2 (Fig. 6A) in NPP1-treated cells. Immunofluorescence staining further revealed that VP treatment in these profibrogenic cholangiocyte-like cells reduces the expression of both YAP1 and COL1 at protein level (Fig. 6B). These results suggest that YAP may play an important role in modulating the fibrotic phenotype of the abnormal cholangiocyte-like cells.

Discussion

In this study, we show that c-Src activity is critical for endodermal commitment of human iPSCs (Fig. 1). We further describe the fate-altering role that Src plays at this stage on the subsequent hepatic specification of iPSC-derived endodermal cells (Figs. 2–3). Transient inhibition of Src during endodermal induction significantly reduces endodermal commitment (Supporting Information Fig. S1), expressing moderate/high levels of pluripotency markers (Fig. 1). The expression of endodermal markers was reduced on treatment with Src kinase inhibitor NPP1 in a dose-dependent manner (Figs. 1–3), whereas high expression of mesodermal markers was seen in endodermal culture conditions (Supporting Information Fig. S3C), promoting a mesodermal fate at the expense of endodermal fate. This initial fate diversion is followed by a progressive cell fate change from the presumptive ensuing HP cell fate to a more cholangiocyte-like fate expressing multiple mature cholangiocyte markers (Fig. 3), but the expression of a few other mature cholangiocyte markers was not significantly increased (Supporting Information Fig. S5). These cholangiocyte-like cells also showed increased expression of fibrosis markers, inflammatory markers, and YAP (Figs. 4, 5). Together, these results show that the cholangiocyte-like cells arising from transient Src inhibition during endodermal commitment of human iPSCs in our study may not be completely functional normal cholangiocytes. Furthermore, as these cells are present with an abnormal phenotype (they are proinflammatory and fibrotic distinctly different from normal cholangiocytes) similar to that seen in biliary diseases such as BA and PSC [37, 38], such a phenotype may actually hamper the complete biliary maturation and functionality. This phenomenon may very well be mimicking the development of the aberrant bile ductal pathology and may reflect a general feature of cholangiocytes observed in disease conditions involving biliary fibrosis [50]. Increased IL-6 has been associated with the infantile liver disease BA [42, 43]. YAP1, in particular, has recently been regarded as a marker of bile duct proliferation and has been reported to increase in many biliary fibrosis conditions including BA, PSC, primary biliary cirrhosis, and bile-duct ligated (BDL) animal models [37, 38]. Evaluation of expression kinetics (Fig. 5) revealed that CK7, COL1, and YAP expression increased from day 5 post Src inhibition during endodermal commitment. Moreover, we observed a significant overlap between COL1 and YAP1 expression (Fig. 5), further augmenting the significance of association of YAP with fibrosis similar to what we have observed in neonatal BA patients' liver [37]. Treatment with VP, a YAP1 inhibitor [37, 48, 49], significantly decreased the expression of markers associated with fibrosis and YAP1 in these profibrogenic cholangiocytes arising from Src inhibition during endodermal commitment (Fig. 6). This also suggests a role that YAP may play in modulating fibrogenesis and give rise to the development of abnormal fibrogenic cholangiocytes. In summary, transient Src inhibition during endodermal commitment of human iPSCs reduced endodermal and negatively influenced further hepatic commitment (Fig. 7). Instead, it promoted a mesodermal fate with early biliary/DP cells arising, which later mature to abnormal cholangiocyte-like cells exhibiting a significant level of fibrogenic potential with expression of a variety of well-known fibrosis markers (Fig. 7).

It has been shown previously that high protein tyrosine kinase activity is essential for early developmental events that precede cell fate specification [51, 52]. Similarly, the activity of SFKs has been shown to be elevated in proliferating pluripotent stem cells [53] and is essential for differentiation of human ESCs [34]. Src inhibition in conjunction with Glycogen synthase kinase 3 (GSK3) inhibition has shown to maintain pluripotency and self-renewal of murine ESCs [54]. Prior research has shown that Src-1 functions in parallel with Wnt signaling to specify endoderm in C. elegans embryos [55]. The presence of c-Src activity alone is sufficient to induce differentiation of murine ESCs to primitive ectoderm and endoderm [31, 56]. In support of the findings from these previous studies, our study further stresses the importance of Src in development and differentiation and suggests a novel critical role of c-Src in human endodermal commitment and the subsequent normal hepatobiliary differentiation. Transient inhibition of Src activity during endodermal commitment of human iPSCs resulted in significant cell fate alteration in endoderm and early hepatic commitment, with a shift toward a cholangiocyte fate. However, these cells express high levels of fibrosis and inflammation markers as well as YAP, a marker associated with ductal proliferation and biliary fibrosis [37, 38, 57]. This may suggest that aberrant Src activity during early endoderm commitment in humans could play a role in the development of biliary fibrosis and neonatal biliary diseases like BA. Although we evaluated the expression of Src in normal, BA, and PSC patient tissues (Supporting Information Fig. S8), we did not observe much difference in its expression, which is not surprising, because such an event (impaired Src activity during early endoderm formation stage) would be an early gestational event, which would not be feasible to study in the human system directly. Although the expression of Src was not significantly different in these human tissues, the protein expression of COL1 and YAP1 in the tissues from patients with biliary diseases including BA and PSC was higher than that observed in the normal control liver (Supporting Information Fig. S8). Interestingly, a significant portion of COL1 positive cells was colocalized with YAP1 or CK7 in the patient tissues (Supporting Information Fig. S8), which is similar to the abnormal proinflammatory fibrotic cholangiocyte-like cells arising from transient Src inhibition during endodermal commitment of human iPSCs (Figs. 5–6). As such, this in vitro human iPSC study may provide potentially important information that cannot be obtained from the developing human fetus. Future studies, however, are warranted to evaluate the in vivo effects of modulating Src during endoderm development in animal models of hepatobiliary development.

Acknowledgments

This work was supported by NIH grants R01EB023812 and R03HD091264, MSCRF, Colleen Mitchel BA 5K, Zachary Meehan Memorial Fund, Johns Hopkins Pediatric Liver Center, and Johns Hopkins Children's Center. Funders had no involvement in study design, interpretation, or decision to publish.

Author Contributions

Y.J.: conception/design, financial support, data analysis and interpretation, manuscript writing; P.C. and L.T.: performed experiments; Q.Z., A.K., R.A., and Z.Y.: provision of study material; P.C., Q.Z., A.K., R.A., Z.Y., S.S., and K.S.: data analysis and interpretation; P.C. and K.S.: manuscript writing.

Disclosure of Potential Conflicts of Interest

K.B.S. declared Consultant/Advisory role with Gilead, Roche, Genentech and research funding Gilead, BMS, Roche, Genentech. The other authors indicated no potential conflicts of interest.

References

Author notes