Isogenic Human Induced Pluripotent Stem Cell Lines Deficient in Biliary Atresia-associated Genes Created by Precise Genome Editing
We targeted both GPC1 and ADD3 to create the panel of isogenic iPSCs based on the highly efficient CRISPR/Cas9 method which we have previously used in human iPSCs from another liver disease (6,16). Two sets of isogenic cell lines, derived from 2 different parental iPSC lines (iHu71 and iBC), were used in this study to achieve more robust/unbiased results. In addition, 3 to 6 replicates of each gene-edited iPSCs were examined for biliary differentiation. Representative data are shown using iHu71 parental and isogenic knock out (KO) lines.
Embryoid Body Differentiation
Embryoid Bodies (EBs) were formed using FBS-containing differentiation medium and cultured in suspension for 7 days. The resulting EBs were then plated on gelatin-coated 24-well plates for additional 3 days. The cells were fixed with 4% paraformaldehyde and stained for markers representing the 3 germ layers.
Immunofluorescence and Flow Cytometry
Human iPSCs and iPSC-derived biliary cells grown on matrigel-coated (Corning) plates were fixed with 4% paraformaldehyde (Sigma) for 20 minutes at room temperature, and washed with phosphate-buffered saline (PBS). Primary antibodies against CK7 (1:200, Cell Marque, Cat. 307M-95), Collagen 1 (1:200, Millipore, Burlington, MA, Cat. 234167), Oct4 (1:200, Millipore, Cat. Mab4401), Nanog (1:200, BD Pharmingen, San Jose, CA, Cat. 560109), Tra160 (1:100, Millipore, Cat. Mab4360), and YAP1 (1:100, Sigma, Cat.wh0010413m1) were diluted in PBS with 0.3% BSA and 0.1% Triton X-100. Fixed cells were incubated overnight with appropriate primary antibodies at 4°C for immunochemistry. The next day, cells were washed twice with PBS and incubated with appropriate Alexa Flour 555 or 488 conjugated secondary antibodies (all of the Alexa Fluor Series from Invitrogen, Carlsbad, CA) in PBS at room temperature for 30 to 45 minutes followed by PBS wash. Cells were then counterstained with DAPI before immunofluorescence analysis. Images were taken using the motorized Nikon Ti-E microscope and NIS-Elements software. For SSEA3 (1:50, Biolegend, Cat. 330306), CK7 (1:400, Cell Marque, Cat. 307M-95), EpCAM (1:200, R&D systems, Minneapolis, MN, Cat. AF960), smooth muscle actin (SMA) (1:1000, Sigma, Cat. A5228) and CK19 (1:100, Santa Cruz, Cat. Sc-6278) flow cytometry analysis, cells were digested by Accutase and washed by PBS. 1 × 105 cells were incubated with Alexa 488-SSEA3 or isotype control antibody for 30 min at 4°C. After PBS washing, the cells were analyzed by a Guava EasyCyte Flow Cytometer (Millipore).
RNA Extraction and Real-time Quantitative Real-time Polymerase Chain Reaction
Total RNA was extracted with TRIZOL reagent (Thermo Fisher, Waltham, MA) according to manufacturer's recommendation. Reverse transcription from mRNA to cDNA was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was used as template for quantitative polymerase chain reaction (Q-PCR) with StepOnePlus Real-Time PCR System (Applied Biosystems), using TaqMan probes. The final PCR reactions consisted of 20 ng cDNA, 1XTaqMan Fast Advanced Master Mix, and TaqMan gene probe at a 20 μL volume. The TaqMan probes included CK7 (Hs00559840_m1), CK19 (Hs00761767_s1), Sox9 (Hs00165814_m1), CFTR (Hs00357011_m1), AE2 (Hs01586776_m1), EpCAM (Hs00901885_m1), Col1 (Hs00164004_m1), SMA (Hs00426835_g1), Loxl2 (Hs00158757_m1), Yap1 (Hs00902712_g1), and 18S rRNA (Hs03003631_g1) in a total volume of 20 μL.
The human iPSC-derived biliary cells were incubated with 100 nM secretin (Sigma, Cat. S7147) in culture media for 20 minutes. The concentration of cAMP was determined using a complete ELISA kit (Abcam, Cat. Ab133051) according to the manufacturer's instruction.
Data is expressed as mean ± standard error of the mean (SEM). For pairwise comparisons, a Student's t test was used. For all tests conducted, P < 0.05 was considered significant.
Altered Biliary Differentiation With Increased Fibrogenesis of Biliary Atresia Patient-specific Induced Pluripotent Stem Cell Lines
Multiple BA patient iPSC lines were compared for their biliary differentiation potential with control iPSCs derived from non-BA controls (Fig. 1). These iPSCs were differentiated into biliary epithelial tissues and cells using the step-wise differentiation protocols (Fig. 1A). We did not observe significant differences between BA and non-BA controls in earlier stages including endoderm, hepatic progenitor stages and the very early stage of biliary differentiation (at ∼day 13, Fig S1 left panel, Supplemental Digital Content, http://links.lww.com/MPG/B508). We did observe biliary defects in the BA cells as early as d15, when small biliary structures normally start to emerge (Fig S1 middle panel, Supplemental Digital Content, http://links.lww.com/MPG/B508). At the end of biliary differentiation (day 20), the BA patient iPSCs showed significantly reduced ductal structure formation in 3D biliary tissue formation assays and did not form detectable levels of ductal cysts/tubes (Fig. 1B, C, and Fig S2, Supplemental Digital Content, http://links.lww.com/MPG/B508). This was consistent with 2D biliary differentiation assays which also showed reduced biliary markers such as CK7, CK19, and EpCAM at protein levels and reduced secretory function in the BA patient iPSCs after biliary differentiation (Fig. 1D, E, F, and Fig S3, Supplemental Digital Content, http://links.lww.com/MPG/B508). Along with the reduced biliary tissue formation, increased fibrosis markers including alpha SMA and Collagen type 1 in the BA patient iPSCs were detected compared to control lines after biliary differentiation of these iPSCs (Fig. 1D, E, G, and Fig S3 http://links.lww.com/MPG/B508). Collagen positive cells are rarely present at any stage of normal biliary differentiation process including the mature stage (Fig. 1G and data not shown). These 2 key disease features, that is, reduced biliary differentiation and increased fibrogenesis, were further confirmed by examining gene expression patterns of more diverse cholangiocyte markers (CK7, CK19, SOX9, CFTR, AE2, and EpCAM) and multiple hepatic fibrosis markers including Collagen type 1, alpha SMA and Loxl2, in diverse iPSC lines derived from 5 different BA patients without anomalies (Fig. 1H). In addition, 1 iPSC line derived from a BA patient with anomalies (midline liver, heterotaxy syndrome with polysplenia), which grows extremely poorly in regular iPSC culture conditions, also showed similar results (ie, reduced biliary marker CK7 and increased fibrosis marker collagen 1 after biliary differentiation, Fig S4, Supplemental Digital Content, http://links.lww.com/MPG/B508).
These data together suggest significantly altered biliary differentiation potential of BA patient iPSCs along with increased fibrosis, recapitulating 2 key clinical features of the disease.
Altered Biliary Differentiation of Gene-edited Human Induced Pluripotent Stem Cell With Specific Defects in Biliary Atresia-associated Genes
Using CRISPR/Cas9, we generated human iPSC lines that have defined mutations in the BA susceptibility loci (GPC1 or ADD3) (Fig. 2A–C). These gene KO iPSC lines were capable of growing similarly to the parental iPSC line and maintained their pluripotency (Fig. 2D). In the biliary differentiation assays, these gene-KO iPSC lines formed significantly decreased amount of biliary tissues in both 3D (over 90% decreased, Fig. 2F) and 2D culture conditions (Fig. 2G, H), and generated increased amount of fibrosis marker positive cells compared to their isogenic control iPSCs at protein and RNA levels (Fig. 2G, H). Similar results were also observed in another set of GPC1 and ADD3 KO iPSCs created from a different parental iPSC (reduced biliary marker CK7 and increased fibrosis marker SMA after biliary differentiation, Fig S5, Supplemental Digital Content, http://links.lww.com/MPG/B508). The degree of the altered biliary differentiation and increased fibrosis were similar in both ADD3 KO lines and GPC1 KO lines. These data show that human iPSCs edited at BA associated genes (GPC1 and ADD3) can recapitulate the key BA disease features including reduced biliary differentiation and increased fibrosis in vitro.
Increased Yes-associated Protein Expression in Biliary Atresia Patient Induced Pluripotent Stem Cells and Knock Out Induced Pluripotent Stem Cells, and the Feasibility of Testing Anti-Fibrotic Drugs in These Human Biliary Atresia Induced Pluripotent Stem Cell Assays
Increased YAP1 expression was detected in all the biliary differentiation cultures of BA patient iPSCs and KO iPSCs at varied levels, compared to control iPSCs (Fig. 3A). In addition, the high nuclear YAP expression was detected in the biliary cells from BA patient-specific iPSCs and interestingly it was well co-localized with collagen type 1 protein (Fig. 3B). Collagen postivie cells are rarely present at any stage of normal biliary differentiation including the mature stage (Fig. 1G, Fig. 3B, and data not shown). We further tested the effect of an anti-fibrogenic drug, pentoxiphylline (36–39), in the biliary differentiation culture of high YAP expressing BA patient-specific iPSCs and KO iPSC (Fig. 3B–E). We observed that this anti-fibrotic drug significantly lowered collagen protein levels as well as decreased the nuclear localization of YAP (Fig. 3B). The levels of collagen 1 and YAP1 gene expression were also decreased in the biliary derivatives of both patient iPSCs and GPC1 KO iPSCs (Fig. 3C, D). In contrast, pentoxiphylline had no effect on those 2 markers (Collagen and YAP) in control iPSCs nor did it alter biliary marker CK7 expression in BA iPSCs (Fig. 3C–E), suggesting the effect was specific to fibrosis in BA rather than affecting normal biliary tissues.
Our study demonstrates the feasibility of generating multiple BA-specific iPSC lines to model human BA in vitro. We further showed that these patient-derived iPSCs resulted in defective biliary differentiation, a key feature of BA, in the in vitro biliary differentiation of patient-iPSC lines; we observed dramatically reduced formation of ductal structures and decreased expression of phenotypic and functional biliary tissue markers at both mRNA and protein levels, compared to their respective controls. Interestingly, increased fibrosis associated markers including collagen 1, alpha SMA and Loxl2 were detected in the BA-patient derived iPSCs, suggesting another key disease feature (ie, increased fibrosis) can be recapitulated from these patient iPSCs. Since the BA patient iPSCs are from humans and recapitulate fibrosis, which was not feasible in the animal models, they hold a great promise as a valuable preclinical in vitro human assay for BA research.
GWAS have identified GPC1 and ADD3, genes that play important roles in embryonic development, as BA susceptibility genes (23–29). Subsequently zebrafish studies have suggested the functional relevance of these genes in impaired intrahepatic biliary network formation (25,28,40). To determine the biological consequences of genetic defects in BA susceptibility genes during human biliary differentiation, we also generated human iPSC lines that have defined mutations in these loci. The functional outcomes of these genetic alterations (GPC1 and ADD3) using human iPSC-based biliary differentiation methods were surprisingly similar to those of BA patient derived iPSCs. In addition, these patient-specific and KO iPSCs also showed increased YAP expression along with increased collagen expression, both of which were lowered with treatment of an anti-fibrotic drug. These results demonstrate that BA disease features can be recapitulated in vitro using precisely gene-edited human iPSCs and provide the feasibility of this approach to determine the functional or pathogenetic roles of GWAS identified genes in this well controlled human biological system; since the isogenic parental control provides the controls with the same genetic backgrounds except the gene of interest. The results obtained in this study also cautiously support the previously less appreciated functional roles of genetic factors in BA pathogenesis. However it is very likely that various other factors including environmental factors trigger BA progression or influence severity of the disease. In fact, a majority of BA patient iPSCs have shown more severe fibrosis phenotypes compared to the KO iPSC lines, indicating the possibility of multiple intrinsic or genetic factors playing roles in BA.
The current study reports the first feasibility of using human iPSC technologies for modeling of key disease features of BA and drug testing in vitro, but this is not a detailed pathogenesis study. Based on these human iPSC-based BA models, our future studies will focus on revealing the pathogenesis underlying each of the disease features, and possible mechanisms whereby GPC1/ADD3 mutations could link to the increased YAP or other common pathways in BA.
Our current study which is heavily focused on developing BA disease models using human iPSCs does not reveal whether the increased collagen expressing cells originated from cholangiocytes or elsewhere. It is not likely that these fibrosis marker positive cells have originated from the mesenchymal cells which are rarely present at any stage of normal biliary differentiation including the early and mature stages (Fig. 1G, Fig 3B, and data not shown). However multiple EMT markers were not significantly increased in the differentiated BA-relevant iPSCs at the mature cholangiocyte stage (Fig S6, Supplemental Digital Content, http://links.lww.com/MPG/B508). Future studies will be needed to dissect if EMT plays a role in earlier differentiation stages when the biliary defect starts to emerge (Fig S1, Supplemental Digital Content, http://links.lww.com/MPG/B508) and identify the cellular origin of BA fibrosis since it could be an important therapeutic target for BA fibrosis prevention/treatment. The phenotypes we observed in the genetically modified iPSCs are similar to those from the BA patients. A limitation of our current study is the absence of direct comparison with primary biliary cells from BA patients since BA patients have no or very limited amount of extrahepatic bile ducts. Another limitation is the lack of complete profiling data comparing the iPSC-cholangiocytes generated from the current differentiation method with human primary cholangiocytes. Their morphology and markers are, however, similar (Fig S7, Supplemental Digital Content, http://links.lww.com/MPG/B508). Therefore, future comprehensive genetic analysis of the patient iPSCs could provide useful insight into the disease mechanisms. The disease models based on patient-specific iPSCs and genetically engineered iPSCs can provide an innovative human-specific platform to validate novel disease targets and to uncover new disease mechanisms. Importantly, the patient-derived iPSC lines can provide a highly human relevant preclinical system for developing or discovery of urgently needed new therapies for BA patients. In addition, this approach can be applied to various other cholangiopathies such as primary sclerosing cholangitis that are currently in need of novel tools to develop effective therapies.
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anti-fibrotic drugs; biliary differentiation; neonatal cholestasis; patient-derived stem cells
Supplemental Digital Content
© 2019 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,